The uses of multiplex detection methodologies are dramatically increasing as a means to increase sample throughput and to demonstrate quantitative differences between multiple targets in gene or protein expression analysis. In this study, we investigate the application of multiplex fluorescent detection for three proteins on the same Western blot using a laser-scanning imaging system, the Bio-Rad Molecular Imager^® FX. We show that independent detection and quantitation of multiple targets is achievable with little or no correction for fluorescent crosstalk by using fluorescent tags preferentially excited with different laser lines and detected at wavelengths that minimize fluorescence crosstalk. We demonstrate that the use of fluorescent detection methods can provide a tenfold greater quantifiable range but with two- to fourfold less sensitivity than chemiluminescent detection methodologies. Two examples of three-color multiplex detection using FITC-, Cy^TM3- and Cy5^TM-conjugated probes on Western blots are provided to demonstrate applications of this approach.