Per- and polyfluoroalkyl substances (PFASs) have been manufactured and used for decades. The aquatic environment is a critical transportation and transformation compartment for PFASs. Target analysis is the most used method of tracing PFASs in the aquatic environment but is powerless for the large amounts of unknown PFASs. This review summarizes the advantages of three target analysis supplementary approaches, including extractable organofluorine (EOF) analysis, high-resolution mass spectrometry (HRMS) screening, and PFAS precursor oxidative conversion. For known PFASs, more targets, including emerging PFASs, PFAS isomers, ultrashort-chain PFASs, and cationic/zwitterionic PFASs, should be considered. For unknown PFASs and organofluorines, developing comprehensive and low-contaminated sample treatment strategies is essential yet challenging. We propose including PFASs screened via HRMS at confidence level (CL) 3 and above in EOF mass balance analysis and CL 4 for samples collected from a “known source”. Appropriate approaches should be applied to investigate unknown PFASs beyond target analysis comprehensively. •Optimizing PFAS treatment method is essential for comprehensive analysis in water.•HRMS-screened PFASs at CLs 1−3 shall be included in EOF mass balance analysis.•Mass-labeled standards shall be used to assess oxidative conversion process.•OF mass balance shall be standardized to facilitate result communication.•Combining HRMS screening and precursor oxidative conversion has broad prospects.