Fluorescence lifetime imaging (FLIM) data has consistently revealed a significant difference in mean FAD lifetime between in vivo and in vitro models. We hypothesized that the observed difference in mean FAD lifetime could be a result of environmental differences, such as differing glucose levels, oxygen levels, or pH, between the two models. We investigated the effects of environmental pH on the autofluorescence lifetime of FAD. We adjusted the pH of HEPEScontaining media using sodium hydroxide and hydrochloric acid. We then replaced the normal media of plated BT474 cells with the pH-adjusted media, allowed 20 minutes for cellular changes to occur, and then imaged the cells using time correlated single photon counting FLIM. We found that the mean lifetime of FAD increased with increased pH, resulting in a significant increase between pH 3.9, 6.2, 7.4, 9.1, and 9.5. The mean lifetime of NAD(P)H decreased at pH 3.9, 9.1, and 9.5 relative to a control pH of 7.3, and the optical redox ratio showed no significant changes except at pH 3.9 relative to a control pH of 7.3. These results suggest that the difference in mean FAD lifetime between in vivo and cell culture models could result from pH changes in the cellular environment.