Numerous studies have found certain unmethylated phytochemicals to possess anti-carcinogenic activity; however, they have been associated with poor oral bioavailability which is a major limiting factor in their usage in chemopreventative treatment. The purpose of this study was to investigate if methylation of a compound would affect bioavailability, in terms of transport and permeability, in a Caco-2 cell model as well as the effect of cell viability and cellular uptake in human colon cancer cell lines. Furthermore, a new analytic method using reversed-phase high performance liquid chromatography coupled with electrochemical detector (HPLC-EC) for the detection and quantification of resveratrol and pterostilbene was developed. This new method was simple, rapid, and more sensitive compared to other detection methods used to analyze resveratrol and pterostilbene. Linear range, limit of detection (LOD), precision and recovery were used to validate this new analytical method. There was a significant increase in intracellular uptake and stronger growth inhibitory of pterostilbene in human cancer cells lines in comparison to resveratrol. Resveratrol exhibited a higher and more rapid rate of transport than pterostilbene across the Caco-2 monolayer regardless of the concentration tested and direction. Pterostiblene exhibited little difference in the rate of transport from either direction. The HCT-116 colon cells had intracellular uptake of each of the polymethoxyflavones (PMFs) tested. Transport was observed by all the PMFs and each had different rates of transport. Overall, location and amount of methyl groups had an effect on bioavailablity of a compound and these compounds show promise as chemopreventative agents.