Results from a study are reported in which patients with leishmaniasis were monitored by whole blood, blood plasma, urine, and hair analysis, before, during, and after intramuscular administration of N-methyl meglumine antimoniate. Quadrupole ICP-MS was used for the detection of antimony and on-line ion chromatography for the separation of its species. After typically 30 consecutive daily injections of 5 mg antimony per kg of body weight, Sb concentrations of up to 250 µg L–1 in whole blood and plasma, and 60 mg of Sb per gram of creatinine in urine, were measured 24 h after drug administration. Antimony in hair samples of these patients showed concentrations of up to 24 µg g–1. Speciation studies of Sb5+ and Sb3+ in drug, urine, and plasma samples were performed by ion chromatography using a Hamilton PRP-100X anion exchange column and EDTA (2 or 20 mM, pH 4.7) as the mobile phases. Repeatability of elution time and peak area measurements for a 0.125 ng spike were <1.2% and <3.5%, respectively. Method detection limits for both species, using a 1:10 diluted urine or plasma sample, were typically 1.6 µg L–1. The procedure was capable of separating the very intense drug peak from its inorganic species, thus permitting the first studies on the bio-transformation of N-methyl meglumine antimoniate to Sb5+ and Sb3+ in the human body.