Background The increasing prevalence of male infertility and the declining trend in sperm quality has been associated to compounds known as “endocrine-disruptors”. The proven endocrine-disrupting effects of atrazine and propazine herbicides led us to conduct long-term research based on highly accurate specific analytical methods with a view to confirming the suspected association. Among the proposed developments was a sensitive analytical method for the simultaneous determination of three metabolites of atrazine and propazine. Results In this work, the method was for first time used for the chromatographic separation and determination of deethyl- and deisopropyl-atrazine (DEA and DIA, respectively) and propazine-2-hydroxy (PP-2OH) in human seminal plasma by LC–ESI-MS/MS using deuterated atrazine (d5-AT) as internal standard (IS). Chromatographic and mass spectrometric conditions such as the mobile phase composition and flow-rate, injected volume, dry gas source temperature and flow-rate, nebulizer pressure and capillary voltage were all carefully optimized. Analytes were identified and quantified by using the multiple reaction monitoring (MRM) mode as applied to positive ions ([M + H]+). Transitions at three different m/z values for each analyte were selected from precursor ions, and the 212.1 → [128]+, 188.1 → [146]+ and 174.1 → [68.1]+ transitions for PP-2OH, DEA and DIA, respectively, were found to be quantitative. The proposed method was validated in terms of precision (repeatability and reproducibility), linear range (10–240 ng mL–1), limit of detection (150–210 pg mL–1), and quantification (500–700 pg mL–1), recovery, accuracy and matrix effects on extracts from variably treated seminal plasma samples. The overall analytical method was successfully applied to human seminal plasma samples from volunteers. PP-2OH was found at concentrations from 1.10 to 11.3 ng mL–1 in four of the six samples, and so was DIA at 9.60 ng mL–1 in one. Conclusions These results are suggestive of bioaccumulation of the target analytes in humans. Untargeted analytes including suspected parent molecules (atrazine and propazine) and other ions [viz., deethyldeisopropyl-atrazine (DD) and diamino-s-chlorotriazine (DACT)] were also detected under the working conditions used. These results may open up new prospects for as yet very incipient research into the bioaccumulation of endocrine disruptors in seminal plasma. Graphical Abstract