We undertook the present study to develop a validated HPLC method for the determination of malondialdehyde (MDA) levels and to use this method for determination of MDA levels in patients with prostate cancer and benign prostatic hyperplasia. MDA levels were estimated in the erythrocyte and plasma sample of the 25 non-metastatic prostate cancer patients, 36 benign prostatic hyperplasia (BPH) patients and 24 age- and sex-matched healthy subjects (controls) in HP Chromatographic systems consisting of a Model Agilent 1100 Series. We report a very rapid and simple isocratic reversed-phase HPLC separation of MDA in normal human plasma and erythrocytes without previous purification of the NMA-TBA complex. All validation parameters were performed in our methods. Using this methods we have found elevated MDA in the plasma and erythrocyte of the prostate cancer group as compared to controls and BPH group. We have improved and validated an analytical HPLC method for determination of MDA in plasma and erythrocyte, which is simple to perform and having high sensitivity, specificity and substantial improvement in column life. This method has been successfully applied to determination of MDA levels in prostate cancer patients and offers an oportunity to further characterize the role of oxidative injury in the pathogenesis of this disease specifically.