Fiber optic probe-based surface plasmon resonance (SPR) detection has been used in combination with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the rapid, sensitive, and selective detection of biomolecules. SPR was used to monitor the covalent immobilization of a polyclonal antibody to the surface of a fiber optic probe. The derivatized probe was then used for the selective detection (from solution) of the corresponding antigen and a secondary antibody directed toward the antigen. Species retained during the SPR detection process were next analyzed by direct MALDI-TOF analysis of the probe surface (after exposed to the MALDI matrix and introduction into the mass spectrometer). The combined approach allowed for the two-dimensional detection of biomolecules, with SPR analysis yielding quantitative information pertinent to the binding events and MALDI-TOF providing details on the qualitative nature of the binding partners.