Non-steroidal anti-inflammatory drugs are drugs with analgesic, antipyretic, and anti-inflammatory effects. This study uses in vitro methods to investigate the potential and unknown genotoxic effects of dexketoprofen trometamol, an active substance in painkillers, on healthy human lymphocytes. In this study, a cytokinesis-block micronucleus cytome assay is used to investigate potential clastogenic, aneugenic activity and to identify chromosome breakages caused by the active drug substance dexketoprofen trometamol; a comet assay is performed to identify the genotoxic damage resulting from DNA single-strand breaks; a real-time reverse transcription polymerase chain reaction panel system is used to evaluate the potential negative effects on the expression of the genes responsible for DNA damage assessment. Dexketoprofen trometamol induces toxic effects in healthy human lymphocytes at concentrations of 750-1000 mu g/mL and above, and shows clastogenic, aneugenic activity by inducing micronucleus formations at exposures of 750-500 mu g/mL. At concentration intervals of 1000, 500, 250, 100 mu g/mL, dexketoprofen trometamol also resulted in DNA damage in the form of strand breaks, as demonstrated by highly significant increases in DNA tail length and density comet parameters when compared to spontaneous values. Human lymphocytes exposed to 750-100 mu g/mL dexketoprofen trometamol were found to have significantly increased levels of expression of the XPC, XRCC6, PNKP genes in the DNA damage signaling pathway. It can be concluded that dexketoprofen trometamol may have cytotoxic, cytostatic, genotoxic effects on healthy human lymphocytes in vitro, depending on the concentration and duration of exposure. It is anticipated that this outcome will be supported by advanced studies.