The present paper deals with the investigations of optimal conditions for the myeloperoxidase (MPO) mediated oxidation of chlorpyrifos, azinphos-methyl and phorate, organophosphorous pesticides (OPs) containing phosphorothionate group, from thio- to oxo-forms, in the presence of hydrogen peroxide. The aim of the work was to apply this oxidation method in the AChE based bioanalytical tests for OPs determination. The maximum concentration of oxo-forms for all tested pesticides was achieved after 10min incubation of OPs in 50mM phosphate buffer (pH 6.0) with 100nM MPO in the presence of 50μM H 2 O 2 . Optimal temperature for obtaining maximal concentration of oxo-forms was 37°C. Only the parent compounds and their oxo-forms were identified chromatographically in the OPs samples after their exposure to MPO. Moreover, no hydrolysis products were detected in the time interval of 1h after the MPO catalyzed reaction was stopped by catalase. The efficiency of OPs transformation from thio- to oxo-forms was measured using acethylcholinesterase (AChE) test, by comparison of percent of AChE inhibition before and after exposure to the oxidized sample.