The GeXP genetic analysis system allows for multiplexed detection and quantitation of up to 35 genes in 192 samples in a single analysis. The analytical procedure includes modified reverse transcription and PCR amplification, followed by capillary electrophoretic separation. RNA material from multiple sample types can be used, including blood, cell lines, and tissue material. This instrumentation has a lower limit of detection of <2 ng with a dynamic range greater than two orders of magnitude, as tested in this validation. Precision experiments demonstrate a within-run coefficient-of-variation (CV) = 11.1% at 25 ng and 12.9% at 12.5 ng total RNA for the complete workflow and CV = 4.8% at 25 ng and 6.7% at 12.5 ng levels for the GeXP analysis alone. The between-run precision for the entire workflow was determined to be 25% at 25 ng. We have devised an optimized protocol and use it to successfully identify a gene expression signature capable of discriminating prostate tumor and non-tumor tissue samples. We used a combination of multiplex gene panels to interrogate ~70 genes in our primary screen. Our results demonstrate that a subset of these genes can be used to separate normal and tumor prostate tissue samples. This protocol using the GeXP analyzer allows for a high-throughput, robust, and reproducible assessment of multiplexed gene expression analysis, and can be used for biomarker discovery to compare different sample groups. With a dynamic linear range and satisfactory precision, this technology holds promise for rapidly identifying gene expression signatures from multiplexed reactions of up to 35 genes in large numbers of samples with limited amounts of starting material.