On-board generation of a set of calibration standards was demonstrated within a microfluidic device designed to perform immunoassay. Electrokinetic flow was used to proportionally mix the antibody (Ab) to bovine serum albumin (BSA) and a diluting buffer, to provide varying Ab concentrations for downstream mixing with fluorescently labeled BSA (BSA*). Mixing ratios were determined from electrical impedance modeling of the fluidic network using P-SPICE software, and peak heights for the labeled species were analyzed relative to the concentration calculated from the model. For dilution and separation of fluorescently labeled amino acids, a linear calibration curve was obtained for mixing ratios of 0.118 to 7.46. A linear calibration curve was obtained for the immunoassay calibration using dilution ratios between 0.197 and 5.077. Deviations were observed at larger extremes, possibly due to leakage effects at intersections. Peak height reproducibility was +/-3% for the immunoassay, using diluted monoclonal Ab in mouse ascites fluid as the analyte. Recovery for on-chip calibration was 92 +/- 6% versus calibrants prepared off-chip, indicating a small bias.