In this study we report depleted uranium (DU) analysis in whole blood samples. Internal exposure to DU causes increased uranium levels as well as change in the uranium isotopic composition in blood specimen. For identification of DU exposure we used the 235U/238U ratio in blood samples, which ranges from 0.00725 for natural uranium to 0.002 for depleted uranium. Uranium quantification and isotopic composition analysis were performed by inductively coupled plasma mass spectrometry. For method validation we used eight spiked blood samples with known uranium concentrations and isotopic composition. The detection limit for quantification was determined to be 4 ng L−1uranium in whole blood. The data reproduced within 1–5% RSD and an accuracy of 1–4%. In order to achieve a 235U/238U ratio range of 0.00698–0.00752% with 99.7% confidence limit a minimum whole blood uranium concentration of 60 ng L¬1 was required. An additional 10 samples from a cohort of veterans exposed to DU in Gulf War I were analyzed with no knowledge of their medical history. The measured 235U/238U ratios in the blood samples were used to identify the presence or absence of DU exposure within this patient group.