Per- and polyfluoroalkyl substances (PFAS) are a large group of environmental contaminants. Humans are exposed to many different PFAS at the same time via food, drinking water, and air. Therefore, evaluation of PFAS mixture effects is an important aspect in PFAS toxicity research. At the molecular level, activation of the peroxisome proliferator-activated receptor alpha (PPARα) has been shown for many PFAS. This transcription factor plays an essential role in lipid metabolism and is regarded as an important molecular target crucial for PFAS toxicity. In the present study, PPARα activation was examined for a number of PFAS mixtures. The mixtures consisted of (M1) classic perfluoroalkyl carboxylic acids (PFCA) and perfluoroalkyl sulfonic acids (PFSA), (M2) novel perfluoroalkyl (poly)-ether carboxylic acids (PFECA) and perfluoroalkyl (poly)-ether sulfonic acids (PFESA), (M3) PFCA and PFECA, and (M4) PFSA and PFESA.