The combination of fluorescence microscopy and time-resolved techniques opens up new possibilities in studying pharmacokinetics and localization of various drugs on a cellular scale. The fluorescence decay time of many molecules is modulated by the physical and chemical properties of the microenvironment, such as viscosity, polarity, pH-value, etc. The alternation of the decay time can also be used to pursue chemical transformations of a drug like degradation, monomerization, or protonation. This paper presents applications of this method from the field of photosensitizing dyes used in photodynamic therapy.