The NanoString nCounter Analysis System uses a digital fluorescent barcode technology that allows for direct multiplexed measurement of gene expression (mRNA), DNA, and protein. The technology uses molecular barcodes and single-molecule imaging to detect and count unique mRNA and protein targets in a single reaction. nCounter-based detection is enzyme-free (no amplification of mRNA is required), fully automated, and allows simultaneous detection of up to 770 mRNA and 30 protein targets from multiple sample types. Target counting is fully digital with quantitative data output. Here we describe preparation of solid tumor lysate samples for use in the nCounter Analysis System.