Fluorescence lifetime (FLT) information is complementary to intensity measurement and can be used to improve signal-to-background contrast and provide environment sensing capability. In this study, we evaluate the FLTs of eight near-infrared fluorescent molecular probes in vitro in various solvent mediums and in vivo to establish the correlation between the in vitro and in vivo results. Compared with other mediums, two exponential fittings of the fluorescence decays of dyes dissolved in aqueous albumin solutions accurately predict the range of FLTs observed in vivo. We further demonstrate that the diffusion of a near-infrared (NIR) reporter from a dye-loaded gel can be detected by FLT change in mice as a model of controlled drug release. The mean FLT of the NIR probe increases as the dye diffuses from the highly polar gel interior to the more lipophilic tissue environment. The two-point analysis demonstrates an efficient in vitro method for screening new NIR fluorescent reporters for use as FLT probes in vivo, thereby minimizing the use of animals for FLT screening studies.