We present a two-dimensional electrophoresis (2DE) method for the detection of the drug, recombinant erythropoietin (rHuEPO) in urine and its separation from endogenous erythropoietin (HuEPO). This method involves a one-step acetonitrile precipitation of the proteins in urine, addition of an internal standard, two-dimensional gel electrophoresis (2D PAGE), a single Western blot and chemiluminescent immunodetection. Results: The 2DE method separates HuEPO and rHuEPO isoforms by both iso-electric point and molecular mass. We have identified several urinary proteins with which the monoclonal EPO antibody used in the current test has non-specific binding. The iso-electric points of these cross-reactive proteins overlap with HuEPO and rHuEPO however, they separate distinctly by the 2DE method. Alpha-2-HS-glycoprotein (HSGP) was identified by peptide mass fingerprinting as one of the urinary cross-reacting proteins, and commercially available purified HSGP was chosen to be added into urine samples as an internal standard prior to separation. Software (EpIQ) was specifically developed that applies four separate criteria to the detection of the migration of rHuEPO and HuEPO relative to the internal standard. Conclusion: The combination of sample preparation, two-dimensional separation, internal standard, standardized blotting procedures and image analysis software enables the 2DE test for rHuEPO in urine to be performed reproducibly and accurately.