In this work, the mechanism of hypericin-induced PDT was investigated in single glioma cells. For this purpose, glioma cells were incubated with hypericin and supplemented with the two additional fluorescence markers resorufin and PI. These three fluorescence markers were chosen as they display distinct FLTs, which allows us to easily distinguish and locate them within a glioma cell. Furthermore, all three markers serve a specific purpose, i.e., hypericin is deployed as photosensitizer for PDT, resorufin is used to label the surrounding medium and PI indicates cellular stress/damage. The combination of all three fluorescence compounds enables us to investigate the individual steps of hypericin-induced PDT on glioma cells and to deduce specific damaging patterns that are indicative of the different programmed cell death mechanisms. We attribute the observed initial cell body shrinkage and bleb formation to be typical features of apoptosis, while the formation of large, non-retracting protuberance, the absence of apoptotic bodies and the explosive membrane rupture were identified as necrotic cell death characteristics. In accordance with control cells, all observed cellular changes during hypericin PDT were proven to be treatment-specific and not to be triggered by laser irradiation. As a result, the cell death occurring in hypericin-induced PDT on glioma cells was found to be primarily apoptosis followed by necrosis. Our study generated new insights and perspectives on hypericin PDT that should contribute to its successful implementation and improvement as an application in brain tumor treatments.