Simultaneous identification and quantification of per- and polyfluoroalkyl substances (PFAS) were evaluated for three quadrupole time-of-flight mass spectrometry (QTOF) acquisition methods. The acquisition methods investigated were MS-Only, all ion fragmentation (All-Ions), and automated tandem mass spectrometry (Auto-MS/MS). Target analytes were the 25 PFAS of US EPA Method 533 and the acquisition methods were evaluated by analyte response, limit of quantification (LOQ), accuracy, precision, and target-suspect screening identification limit (IL). PFAS LOQs were consistent across acquisition methods, with individual PFAS LOQs within an order of magnitude. The mean and range for MS-Only, All-Ions, and Auto-MS/MS are 1.3 (0.34–5.1), 2.1 (0.49–5.1), and 1.5 (0.20–5.1) pg on column. For fast data processing and tentative identification with lower confidence, MS-Only is recommended; however, this can lead to false-positives. Where high-confidence identification, structural characterisation, and quantification are desired, Auto-MS/MS is recommended; however, cycle time should be considered where many compounds are anticipated to be present. For comprehensive screening workflows and sample archiving, All-Ions is recommended, facilitating both quantification and retrospective analysis. This study validated HRMS acquisition approaches for quantification (based upon precursor data) and exploration of identification workflows for a range of PFAS compounds.