This study deals with the investigation of hypericin PDT on single, living glioma cells by means of spatially resolved fluorescence spectroscopy and fluorescence lifetime imaging microscopy (FLIM). An introduction to the acquisition and analysis of fluorescence lifetime data is given in section 1 of the supporting information (SI). Three fluorescent markers: hypericin, resorufin and propidium iodide (PI), were chosen to initiate and monitor hypericin-induced PDT. The distinct fluorescence lifetimes (FLTs) of these dyes, in conjunction with spatially resolved fluorescence spectroscopy, enables monitoring their respective localization and photophysical behavior during the progression of the PDT. This allows imaging the photodamaging progression and identify the underlying cell death mechanism.