Display omitted •Photoactivation process of Erythrosine was investigated in different simulated body fluids.•Optical techniques were used to investigate the photoactivation process.•Thermal lens results reveled photobleaching in submicromolar concentrations range.•Clear differences in the photodegradation kinetics, triplet lifetime and singlet oxygen generation were observed.•The results presented could help to explain and to define Photodynamic Therapy application protocols. Photodynamic Therapy (PDT) is a powerful technique for the treatment of cancer and non-cancerous diseases. The precise PDT treatment protocol definition must consider the performance difference between in vitroand in vivoapplications. This also occurs in other biological studies, and to partially overcome this difficulty, the simulated body fluids are generally applied as a prior understanding of the particularities of the different systems. However, in PDT these studies are scarce. In this work, we investigated the photoactivation of Erythrosine, a photosensitizer widely used in PDT, in different simulated body fluids. Differences in the photodegradation kinetics, triplet lifetime, and singlet oxygen generation were observed. The results can help to explain and to define PDT application protocols.