Organofluorines occur in human serum as complex mixtures of known and unidentified compounds. Human biomonitoring traditionally uses targeted analysis to measure the presence of known and quantifiable per- and polyfluoroalkyl substances (PFAS) in serum, yet characterization of exposure to and quantification of PFAS are limited by the availability of methods and analytical standards. Studies comparing extractable organofluorine (EOF) in serum to measured PFAS using organofluorine mass balance show that measurable PFAS only explain a fraction of EOF in human serum and that other sources of organofluorine may exist. The gap in fluorine mass balance has important implications for human biomonitoring because the total body burden of PFAS cannot be characterized and the chemical species that make up unidentified EOF are unknown. Many highly prescribed pharmaceuticals contain organofluorine (e.g., Lipitor, Prozac) and are prescribed with dosing regimens designed to maintain a therapeutic range of concentrations in serum. Therefore, we hypothesize organofluorine pharmaceuticals contribute to EOF in serum. We use combustion ion chromatography to measure EOF in commercial serum from U.S. blood donors. Using fluorine mass balance, we assess differences in unexplained organofluorine (UOF) associated with pharmaceutical use and compare them with concentrations of organofluorine predicted based on the pharmacokinetic properties of each drug. Pharmacokinetic estimates of organofluorine attributable to pharmaceuticals ranged from 0.1 to 55.6 ng F/mL. Analysis of 44 target PFAS and EOF in samples of commercial serum (n = 20) shows the fraction of EOF not explained by Σ44PFAS ranged from 15% to 86%. Self-reported use of organofluorine pharmaceuticals is associated with a 0.36 ng F/mL (95% CL: −1.26 to 1.97) increase in UOF, on average, compared to those who report not taking organofluorine pharmaceuticals. Our study is the first to assess sources of UOF in U.S. serum and examine whether organofluorine pharmaceuticals contribute to EOF. Discrepancies between pharmacokinetic estimates and EOF may be partly explained by differences in analytical measurements. Future analyses using EOF should consider multiple extraction methods to include cations and zwitterions. Whether organofluorine pharmaceuticals are classified as PFAS depends on the definition of PFAS.