This talk will present recent developments at the University of New Mexico in the development of microfluidic based biosensor systems and their use in analysis of biomolecular recognition. The method involves real-time detection of soluble molecules binding to receptor-bearing microspheres, sequestered in affinity column-format inside a microfluidic channel. The packed microcolumn format is (1) well suited for enhancing reaction times of analyte with immobilized receptors, (2) compatible with electro-osmotic pumping, and (3) allows detection of multiple analytes. Identification and quantitation of analytes occurs via direct fluorescence measurements or fluorescence resonance energy transfer (FRET). Several immunoassays have been developed that can potentially detect sub-femtomole quantities of antibody with high signal-to-noise ratio and a large dynamic range spanning nearly four orders of magnitude in analyte concentration in microliter to submicroliter volumes of analyte fluid. Kinetic and equilibrium constants for the reaction of this receptor-ligand pair are obtained through modelling of kinetic responses of the affinity microcolumn and are consistent with those obtained by flow cytometry. Because of the correlation between kinetic and equilibrium data obtained for the microcolumns, quantitative analysis can be done prior to the steady state endpoint of the recognition reaction. This method has the promise of combining the utility of affinity chromatography, with the advantage of direct, quantitative, and real-time analysis and the cost-effectiveness of microanalytical devices. The approach has the potential to be generalized for high sensitivity, high selectivity, rapid detection of a host of bioaffinity assay methods and analyte types.