Therapeutic recombinant-methionyl human leptin (r-metHu-Leptin, Mr 16155) shares an identical amino acid sequence with endogenous leptin (endo-leptin, Mr 16024), with the addition of an N-terminal methionyl incorporated during recombinant expression in Escherichia coli. Current immunochemistry-based assays do not allow discrimination between the drug and endo-leptin because of cross reactivity. Using the immunoassay, the total plasma concentration measured in some clinical study subjects receiving r-metHu-Leptin can reach supra-physiological levels. To determine which leptin species contributes to the elevated concentrations detected in some subjects, a mass spectrometry-based method allowing discrimination and quantification of both leptin species was developed.Endo-leptin and r-metHu-Leptin were enriched from plasma matrix proteins by immuno capture, and subsequently injected onto a reversed phase analytical column coupled to an API 4000 Q-TRAP LC–MS/MS system. Multiple charge state ions and specific MRMs were monitored to provide unambiguous differentiation between endo-leptin and r-metHu-Leptin.A “top down” assay distinguishing the two forms of leptin was successfully developed and had a linear range from 15.63 to 1000ng/ml, low limit of quantification of 15.63ng/ml. The method was applied to selected clinical samples and revealed that the elevated leptin concentrations observed in some subjects reflected accumulation of r-metHu-Leptin.An LC–MS/MS method was developed for unambiguous differentiation of r-metHu-Leptin from endogenous leptin in human plasma. Using this method, specific quantitative information was obtained for pharmacokinetic studies in a clinical trial. The method should prove useful in quantifying this investigational drug against endo-leptin background in future clinical studies.