Single- and multi-analyte detection of two gonadotropic hormones (follicle stimulating hormone (hFSH) and luteinizing hormone (hLH)) was achieved by a Surface Plasmon Resonance (SPR) immunoassay on untreated human urine samples. Multi-analyte detection was accomplished using two alternative formats which are based in the individual or simultaneous immobilization of the hormones on the sensor surface. The lowest detection limit for both hormones in urine was found to be 1 ng mL(-1), which in international units (IU) in terms of the World Health Organization (WHO) standards represents 8 mIU mL(-1) of hLH and 14 mIU mL(-1) of hFSH, respectively. The reliability of the assay was demonstrated by intra- and inter-assay variabilities < 6%, chip-to-chip variabilities < 5%, recoveries in the range of 80-120% and stability of the sensor response through more than 100 measurements. The sensitivity of this biosensing methodology renders it in a useful technique for the diagnosis of reproductive disorders, as well as for fertility monitoring.