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Abstract Land application of treated sewage sludge (also known as biosolids) is considered a sustainable route of disposal because it reduces waste loading into landfills while improving soil health. However, this waste management practice can introduce contaminants from biosolids, such as per- and polyfluoroalkyl substances (PFAS), into the environment. PFAS have been observed to be taken up by plants, accumulate in humans and animals, and have been linked to various negative health effects. There is limited information on the nature and amounts of PFAS introduced from biosolids that have undergone different treatment processes. Therefore, this study developed analytical techniques to improve the characterization of PFAS in complex biosolid samples. Different clean-up techniques were evaluated and applied to waste-activated sludge (WAS) and lime-stabilized primary solids (PS) prior to targeted analysis and suspect screening of biosolid samples. Using liquid chromatography with high-resolution mass spectrometry, a workflow was developed to achieve parallel quantitative targeted analysis and qualitative suspect screening. This study found that concentrations of individual PFAS (27 targeted analytes) can range from 0.6 to 84.6 ng/g in WAS (average total PFAS = 241.4 ng/g) and from 1.6 to 33.8 ng/g in PS (average total PFAS = 72.1 ng/g). The suspect screening workflow identified seven additional PFAS in the biosolid samples, five of which have not been previously reported in environmental samples. Some of the newly identified compounds are a short-chain polyfluorinated carboxylate (a PFOS replacement), a diphosphate ester (a PFOA precursor), a possible transformation product of carboxylate PFAS, and an imidohydrazide which contains a sulfonate and benzene ring.