AbstractNext‐generation sequencing offers many advantages over other methods of microRNA (miRNA) expression profiling, such as sample throughput and the capability to discover novel miRNAs. As the sequencing depth of current sequencing platforms exceeds what is necessary to quantify miRNAs, multiplexing several samples in one sequencing run offers a significant cost advantage. Although previous studies have achieved this goal by adding bar codes to miRNA libraries at the ligation step, this was recently shown to introduce significant bias into the miRNA expression data. This bias can be avoided, however, by bar coding the miRNA libraries at the PCR step instead. Here, we describe a user‐friendly PCR bar‐coding method of preparing multiplexed microRNA libraries for Illumina‐based sequencing. The method also prevents the production of adapter dimers and can be completed in one day. Curr. Protoc. Hum. Genet. 73:11.12.1‐11.12.10 © 2012 by John Wiley & Sons, Inc.