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Although historic perfluorinated compounds are currently under scrutiny and growing regulatory control in the world, little is known about human exposure to other polyfluorinated compounds presently in use. Fluorotelomer alcohols (FTOHs) and polyfluoroalkyl phosphate esters (PAPs) are known to degrade to terminal perfluorinated acids and toxic reactive intermediates through metabolic pathways. Therefore, it is important to characterize their human exposure by the identification of unique biomarkers. With the use of liquid chromatography mass spectrometry-time-of-flight analysis (LC-MS-TOF), we developed a workflow for the identification of metabolites for the 8:2 FTOH and 8:2 diPAP. Analysis of serum and urine of dosed rats indicated the 8:2 FTOH sulfate and the 8:2 diPAP as potential biomarkers. These compounds, as well as 25 other fluorinated compounds and metabolites, were analyzed in human serum and urine samples from the general population (n = 100) and office workers (n = 30). The 8:2 FTOH sulfate was measured for the first time in human samples in 5 to 10% of the serum samples, ranging from 50 to 80 pg/mL. The 8:2 diPAP was measured in 58% of the samples, ranging from 100 to 800 pg/mL. This study indicates the FTOH sulfate conjugate as a biomarker of exposure to FTOHs and PAPs in humans.