The presence of unknown organofluorine compounds in environmental samples has prompted the development of nontargeted analytical methods capable of detecting new perfluoroalkyl and polyfluoroalkyl substances (PFASs). By combining high volume injection with high performance liquid chromatography (HPLC) and ultrahigh resolution Orbitrap mass spectrometry, a sensitive (0.003-0.2 ng F/mL for model mass-labeled PFASs) untargeted workflow was developed for discovery and characterization of novel PFASs in water. In the first step, up to 5 mL of water is injected to in-line solid phase extraction, chromatographed by HPLC, and detected by electrospray ionization with mass spectral acquisition in parallel modes cycling back and forth: (i) full scan with ultrahigh resolving power (RP = 120,000, mass accuracy ≤3 ppm), and (ii) in-source fragmentation flagging scans designed to yield marker fragment ions including [C2F5](-) (m/z 118.992), [C3F7](-) (m/z 168.988), [SO4H](-) (m/z 96.959), and [Cl](-) (m/z 34.9). For flagged PFASs, plausible empirical formulas were generated from accurate masses, isotopic patterns, and fragment ions. In the second step, another injection is made to collect high resolution MS/MS spectra of suspect PFAS ions, allowing further confirmation of empirical formulas while also enabling preliminary structural characterization. The method was validated by applying it to an industrial wastewater, and 36 new PFASs were discovered. Of these, 26 were confidently assigned to 3 new PFAS classes that have not previously been reported in the environment: polyfluorinated sulfates (CnFn+3Hn-2SO4(-); n = 5, 7, 9, 11, 13, and 15), chlorine substituted perfluorocarboxylates (ClCnF2nCO2(-); n = 4-11), and hydro substituted perfluorocarboxylates (HCnF2nCO2(-); n = 5-16). Application of the technique to environmental water samples is now warranted.