Microchip frontal affinity chromatography was demonstrated to estimate the binding of 5-carboxyfluorescin-(D-Ala)(3) (1) to magnetic microbeads derivatized with teicoplanin (Teic) from Actinoplanes teicomyceticus. In this technique, a cross-chip was used whereby the two side channels contained an identical length (1.5 mm) of derivatized Teic microbeads (affinity column) and underivatized beads (control column), respectively. Cylindrical NdFeB magnets were fabricated into the PDMS chips to retain the magnetic beads. Upon application of a voltage, a sample of 1 was continuously introduced into the affinity column followed by a buffer wash and the same sample from the control channel. The extent of interaction between 1 and the two types of beads in either microchannel resulted in differences in migration time of the ligand as detected by fluorescence. This difference was used to obtain a value for the binding constant between 1 and Teic-beads of 5.4 x 10(4) M-1. This technique reduces the amount of sample needed for the binding assay as compared with conventional frontal affinity chromatography techniques.