Nanomaterial-based delivery systems have been increasingly employed in nanomedicine to improve targeting and enhance therapeutic efficacy. Understanding how nanomaterials behave in vivo is essential for optimizing the delivery of therapeutic cargos or drugs. This dissertation utilizes laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP-MSI) to investigate the fate of nanomaterials in biological systems. LA-ICP-MSI offers high sensitivity, multiplexing capability, site-specific information, and absolute quantification of nanomaterials in vivo. However, the technique faces limitations such as the lack of matrix-matched standards, signal drift caused by instrumental fluctuations, and relatively low spatial resolution. To overcome these limitations, a tissue-mimicking approach was developed to better replicate the properties of biological tissues. When combined with an internal standard spiked into gelatin, this method enables more accurate absolute quantification of nanomaterials in biological samples. For instance, it was used to analyze the distribution of gold nanoparticles (AuNPs) in spleen tissue at one and six days after intravenous administration, revealing their interaction with the immune system. The method was also applied to evaluate the colocalization of nanozyme components, including AuNPs and a palladium catalyst, in tumors. Spatial mapping confirmed that both components remained intact in vivo. This dissertation also demonstrates the multiplexed tracking of proteins delivered by polymeric nanocarriers. By integrating metal-coded mass tags (MMTs) with ICP-MS and LA-ICP-MSI, this approach enables the quantification and spatial localization of multiple proteins delivered by polymers in a multiplexed fashion. This capability allows for the identification and optimization of nano-delivery systems better suited for targeting specific cells or disease sites, while minimizing off-target effects. Furthermore, improving the spatial resolution of LA-ICP-MSI is shown to be critical for investigating nanomaterial distribution within sub-organ regions. Computational methods were employed to enhance spatial resolution to approximately 5 µm, allowing for detailed assessment of nanomaterials in small anatomical structures. This capability was demonstrated by tracking AuNPs in sub-organ regions of the spleen and liver. In summary, this dissertation highlights the value of LA-ICP-MSI in studying the in vivo behavior of nanomaterials. The insights gained from this work are valuable for advancing drug delivery research and developing more effective nanomedicine strategies.