Verteporfin and Lemuteporfin are compared to examine the effect of their functional groups and therefore the localization in two-photon excitation (TPE) photodynamic therapy (PDT). We used singlet oxygen-related photobleaching of the sensitizers to assess TPE-induced singlet oxygen generation in multilamellar vesicles (MLVs) and U343 glioma cells under a variety of conditions. It was found that Lemuteporfin photobleached at a faster rate than Verteporfin in the majority of environments. Also, Verteporfin and Lemuteporfin exhibited different behaviors when in hypoxic environments relative to those in oxygenated MLVs. These differences are attributed to the sensitizer location in the membrane and their relative mobilities throughout membranes and cells.