Standardized RT-PCR (StaRT-PCR) enables numerical quantification as well as intra- and inter-laboratory comparison of gene expression. Multiplex StaRT-PCR, using two rounds of amplification, was conducted on Stratagene Universal Reference RNA. In the first round, cDNA, competitive template (CT) mix, and primers for up to 96 genes were amplified for varying numbers of cycles. Next, products from round one were diluted, combined with primers for one gene, and amplified for an additional 35 cycles. No additional cDNA or CT mix was added. Expression values obtained by uniplex and multiplex StaRT-PCRs were highly correlated (R=0.993, p<0.001). Products from round one could be diluted as much as 100,000-fold and still be quantified following round two amplification. Thus, using multiplex StaRT-PCR, 96 genes were measured in the same amount of cDNA typically used to measure one gene with uniplex StaRT-PCR. Multiplex StaRT-PCR was also used to measure 18 genes in the fine needle biopsy of a primary lung carcinoma.