This unit presents the protocol for the NanoString nCounter Gene Expression Assay, a robust and highly reproducible method for detecting the expression of up to 800 genes in a single reaction with high sensitivity and linearity across a broad range of expression levels. The methodology serves to bridge the gap between genome‐wide (microarrays) and targeted (real‐time quantitative PCR) expression profiling. The nCounter assay is based on direct digital detection of mRNA molecules of interest using target‐specific, color‐coded probe pairs. It does not require the conversion of mRNA to cDNA by reverse transcription or the amplification of the resulting cDNA by PCR. Each target gene of interest is detected using a pair of reporter and capture probes carrying 35‐ to 50‐base target‐specific sequences. In addition, each reporter probe carries a unique color code at the 5′ end that enables the molecular barcoding of the genes of interest, while the capture probes all carry a biotin label at the 3′ end that provides a molecular handle for attachment of target genes to facilitate downstream digital detection. After solution‐phase hybridization between target mRNA and reporter‐capture probe pairs, excess probes are removed and the probe/target complexes are aligned and immobilized in the nCounter cartridge, which is then placed in a digital analyzer for image acquisition and data processing. Hundreds of thousands of color codes designating mRNA targets of interest are directly imaged on the surface of the cartridge. The expression level of a gene is measured by counting the number of times the color‐coded barcode for that gene is detected, and the barcode counts are then tabulated. Curr. Protoc. Mol. Biol. 94:25B.10.1‐25B.10.17. © 2011 by John Wiley & Sons, Inc.