This bifunctional biotin derivative is used for spectrophotometric determination of biotinylation degree. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The fluorescence enhancement of fluorescein biotin is proportional to the concentration of biotin. Because the assay is amenable for use in small volumes of 5-50 uL or bead-based assays, the detection limits can be extended to the femtomole range. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.