Per- and polyfluoroalkyl substances (PFASs) are among the persistent organic pollutants characterized by their persistence in the environment, high mobility, and adverse impact not only on the ecosystem but also on human health. The biggest challenges in human biomonitoring are the low concentrations of PFASs in biological matrices and the presence of matrix interferents in samples. The combination of liquid chromatography with tandem mass spectrometry (LC-MS/MS) and solid-phase extraction (SPE) as a sample preparation technique appears to be the most suitable solution for achieving the desired selectivity and sensitivity in PFAS determination. The aim of this review is to describe possible sources of PFASs, their presence in various human matrices, analytical methods for determining PFASs in different biological matrices using various pretreatment techniques for complex samples, as well as adverse health risks associated with PFAS exposure. The most studied PFASs include PFOA and PFOS, which are most frequently detected in matrices such as plasma, serum, and breast milk. The average concentrations of PFOA range from 1.0 to 2.6 ng.mL in plasma, 1.9 to 2.4 ng.mL in serum, and 0.4 to 3.1 ng.mL in breast milk. For PFOS, the average concentrations were 2.0-4.0 ng.mL, 3.7-4.6 ng.mL, and 3.6-4.8 ng.mL for plasma, serum, and breast milk, respectively. The most significant health effects associated with exposure to long-chain PFASs (such as PFOA and PFOS) include lipid disorders, hypertension, diabetes mellitus, thyroid disorders, infertility, cancer, obesity, autism, neurodevelopmental issues, cardiovascular diseases, and kidney and liver disorders. It is of utmost importance to monitor PFAS exposure, predict their toxicity, and develop effective strategies to mitigate their potential effects on human health.