OBJECTIVE Our study aims to assess the oxidative stress status of seminal plasma from normozoospermic, azoospermic, and leukocytospermic males, since abnormal sperm and leukocytes in human ejaculates are the main source of reactive oxygen species (ROS) which lead to oxidative damages. For this purpose we applied a biochemical approach to the assessment of the oxidative stress status by using two-dimensional (2D) electrophoresis to check the level of protein oxidation after specific labeling of free thiol (-SH) groups. METHODS Seminal plasma samples from normal and pathological males were analyzed by a luminol-based chemiluminescent assay. The same samples after specific labeling of free -SH groups with 3-N-maleimidopropionyl biocytin, were analyzed by 2D electrophoresis and computer-assisted semiquantitative determination of the amount of free -SH groups. RESULTS Using a standard chemiluminescence assay, we demonstrated a high, low and normal level of ROS, respectively, in seminal plasma from leukocytospermic, azoospermic, and normozoospermic subjects. By 2D electrophoresis and streptavidin blotting of specifically labeled free -SH groups of proteins, we detected in the same samples a higher level of oxidated -SH groups comparable between azoospermic and leukocytospermic samples, whereas a significantly higher level of free -SH groups was detected in normozoospermic subjects. CONCLUSIONS Our results demonstrated that a pathological oxidative stress status in seminal plasma may be revealed by the levels of the protein free -SH groups, both in the presence or absence of cells.