Wastewater-based epidemiology (WBE) started to develop significantly in the last 8 years due, in part, to the advance of liquid chromatography–mass spectrometry (LC–MS). Its initial objective ― to estimate the consumption of illicit drugs ― has been expanded to new psychoactive substances (NPS) and other human biomarkers of health, lifestyle and disease. The purpose of this review is to introduce recent methods and outstanding challenges in the application of LC–MS to these new targets, including target, suspect and non-target screening and, outline the workflows developed for discovering metabolites of NPS and biomarkers. Further, we discuss a number of specific methodological challenges that also deserve scrutiny in future research.