Circadian rhythms are physical and behavioral changes that follow a daily light-dark cycle over 24 hours. They are crucial in regulating several physical and cellular processes. Epidemiological studies have linked alterations in circadian rhythms (such as light at night, social jet lag, night shift work, etc.) to multiple diseases, including metabolic, cardiovascular, neurodegenerative diseases, and cancer. On the cellular level, circadian rhythms are controlled by a molecular clock, a network of genes/proteins whose transcription and translation oscillate, regulating cellular pathways. This dissertation focuses on chemical biology-mediated studies of circadian rhythms in vitro. In Chapter 2, we evaluated the effects of melatonin on PER2 and BMAL1 oscillations in U2OS cells. Melatonin is a key regulator of the suprachiasmatic nucleus (SCN) and binds to melatonin receptor 1 (MT1), a G-coupled protein receptor found on cells in the SCN and peripheral tissue. Therefore, it is important to understand the effects of melatonin on circadian gene oscillations in cells derived from peripheral tissue. We assessed the effects of melatonin and an MT1 inverse agonist, UCSF7447, in U2OS circadian reporter cell lines. We found that both melatonin and UCSF7447 have period-lengthening effects on cellular oscillations, illustrating that the activity of these molecules is not entirely dependent upon the SCN. Melatonin delayed circadian phases while UCSF7447 advanced them, corroborating in vivo data. We also studied how dosing times of these compounds affect circadian oscillations. Following delayed treatments, we did not observe any differences in the impact of these molecules on the oscillations. Our findings demonstrate the importance of utilizing in vitro models and that the direct effects of melatonin can go beyond the SCN and should be explored further. In Chapter 3, we generated a SNAIL:luciferase promoter-reporter to track SNAIL transcription in U2OS, MCF10A, MCF7, and MDA-MB-231 cells. We expanded on previous work done by the group, which found that while BMAL1 and PER2 transcription oscillate in normal breast (MCF10A) cells, cells that model breast cancer had dampened or no oscillations (MCF7 and MDA-MB-231, respectively). SNAIL regulates the epithelial-to-mesenchymal transition, which is important in embryonic development and wound healing in adults. However, in breast cancer, SNAIL is linked to tumor progression and recurrence. Prior studies have suggested that SNAIL has a circadian expression pattern. Western blots and real-time quantitative polymerase chain reactions (RT-qPCRs) were utilized for these studies, which are low resolution compared to luciferase reporters. We report that SNAIL transcription oscillates in U2OS cells. Also, initial experiments indicate that SNAIL transcription oscillates in MCF10A and MCF7 cells. Meanwhile, MDA-MB-231 cells, which represent an aggressive breast cancer phenotype, do not have any SNAIL oscillations. In Chapter 4, we incorporated an auxin-inducible degron into U2OS cells to selectively degrade PER2, CRY1, and CRY2. Genetic knockouts and knockdowns are used to perturb protein function. While genetic knockouts and knockdowns have been pivotal in our understanding of the circadian machinery in cells, we are interested in directly studying circadian proteins since they are the functional elements of the circadian loop. While small molecules are utilized to perturb circadian proteins, most of the molecules that exist bind to stabilize CRY or bind to secondary loop proteins, ROR, and REV-ERB. Due to the difficulty in generating new molecules, we used an alternative method to degrade endogenous circadian proteins, an auxin-inducible degron (AID). We knocked the degron into the terminus of PER2, CRY1, or CRY1 via CRISPR Cas9, but we could not degrade our target proteins. In addition to describing the design and generation of the constructs, we propose ways to fix and troubleshoot the degradation. We also want to compare the protein degradation model to knockout and knockdown models to better understand the circadian machinery across the central dogma. In Appendix A, we present data on the changes in mRNA and protein levels of PER and CRY after PER2, CRY1, and CRY2 knockdown. We also assessed mRNA levels of PER1 and PER2 in a PER2 knockout model. Our results indicate that knockdown of PER2, CRY1, and CRY2 siRNA resulted in reduced mRNA levels of these genes, but we only observe compensatory upregulation for the CRY1/2 isoforms, not PER1/2. At the protein level, knockdown of CRY2 mRNA resulted in a reduction in CRY2 protein, while no decrease in protein levels was observed following PER2 or CRY1 knockdown. Furthermore, no protein-level compensation was detected in any of these models. In the PER2 knockout model, PER2 mRNA levels were reduced, but PER1 levels remained unchanged.