Display omitted •A new LC-QTOF-MS method for 4 PDE5Is was developed and validated.•49 food supplements for enhancing sexual performance were analyzed.•65% of samples contained PDE5Is and their structural analogs.•2 PDE5Is and 8 PDE5Is analogs were identified in food supplements.•Undeclared PDE5Is and their analogs in food supplements pose risks to human health. This paper presents the development and validation of an analytical method using liquid chromatography-time of flight mass spectrometry (LC-QTOF-MS) suitable for screening and identifying suspect PDE5I analogs and quantifying four target PDE5Is in various food supplements. The method validation comprised of selectivity, linearity, accuracy, the limit of detection (LOD) and quantification (LOQ), and evaluation of matrix effects. The method showed good linearity with coefficients of determination higher than 0.992 for all analytes. LODs and LOQs ranged from 0.2 to 2.2 ng/mL and 2 to 20 ng/mL, respectively. The accuracy and precision of the method were deemed satisfactory, with values ranging from 88.5 to 115.9 % for intra- and 86.9 to 111.2 % for inter-day experiments, and RSDs ranged from 0.4 to 4.5 % and 0.9 to 5.2 %, respectively. Out of 49 analyzed food supplements collected on the Croatian market, 65 % were not in compliance with EU regulations and pose possible risks to human health.