Perfluoroalkyl substances (PFASs) are proliferated into the environment on a global scale and present in the organisms of animals and humans even in remote locations. Persistent organic pollutants of that kind therefore have stimulated substantial improvement in analytical methods. The aim of this review is to present recent achievements in PFASs determination in various matrices with different methods and its comparison to measurements of Total Organic Fluorine (TOF). Analytical methods used for PFASs determinations are dominated by chromatography, mostly in combination with mass spectrometric detection. However, HPLC may be also hyphenated with conductivity or fluorimetric detection, and gas chromatography may be combined with flame ionization or electron capture detection. The presence of a large number of PFASs species in environmental and biological samples necessitates parallel attempts to develop a total PFASs index that reflects the total content of PFASs in various matrices. Increasing attention is currently paid to the determination of branched isomers of PFASs, and their determination in food.FigureThe aim of this review is to present recent achievements in perfluoroalkyl substances (PFASs) determination in various matrices with different methods and its comparison to measurements of Total Organic Fluorine (TOF). Increasing attention is currently paid to the determination of branched isomers of PFASs, and their determination in food.