The accurate determination of perfluoroalkyl substances (PFSAs) in water, sediment, fish, meat, and human milk was achieved by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QqTOF-MS) with an ABSciex Triple TOF®. A group of 21 PFSAs was selected as target to evaluate the quantitative possibilities. Full scan MS acquisition data allows quantification at relevant low levels (0.1-50 ng L(-1) in water, 0.05-2 ng g(-1) in sediments, 0.01-5 ng g(-1) in fish and meat, and 0.005-2 ng g(-1) in human milk depending on the compound). Automatic information dependent acquisition product ion mass spectrometry (IDA-MS/MS) confirms the identity even for those compounds that presented only one product ion. The preparation of a homemade database using the extracted ion chromatogram (XIC) Manager of the software based upon retention time, accurate mass, isotopic pattern, and MS/MS library searching achieves not only the successful identification of PFSAs but also of some pharmaceuticals, such as acetaminophen, ibuprofen, salicylic acid, and gemfibrozid. Mean recoveries and relative standard deviation (RSD) were 67-99% (9-16% RSD) for water, 62-103% (8-18% RSD) for sediment, 60-95% (8-17% RSD) for fish, 64-95% (8-15% RSD) for meat, and 63-95% (8-16%) for human milk. The quantitative data obtained for 60 samples by UHPLC-QqTOF-MS agree with those obtained by LC-MS/MS with a triple quadrupole (QqQ).