The proper design of experiments is a critical step for each study in order to obtain reproducible and reliable data. Taking into account constant competitiveness in the quickly developing biomedical sciences and the availability of sophisticated techniques, the choice and establishment of an experimental model system are essential for a successful research project. Currently, various sophisticated in vitro and in vivo models are being designed and developed in order to replace the use of mammalian models to investigate the mechanisms of action, activity and properties of novel compounds or treatment modalities. After the clinical success of photodynamic therapy (PDT) used against neovascular eye disorders, clinically approved PDT protocols for cancer are still being developed. Since several aspects of PDT should be examined, it is crucial to define the possible models that would guide the principle of the 3Rs (Reduction, Refinement, Replacement) practice, which is known as the fundamental strategy for designing more ethical animal studies. This review is focused on the usefulness of alternative in vivo as well as in vitro models to study important aspects of PDT, especially in the context of cancer research. 3D human-relevant cell culture models, followed by non-mammalian models such as the nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), zebrafish (Danio rerio), frog (Xenopus laevis), or chicken chorioallantoic membrane (Gallus gallus), are discussed as an alternative to the widely used but ethically controversial mammalian models.