This protocol describes how to isolate exosomal RNA from serum/plasma by using Millipore membrane filters for exosomal enrichment followed by the Qiagen miRNeasy Micro kit for RNA isolation. It supercedes an earlier protocol (http://dx.doi.org/10.1038/protex.2015.110). The new protocol is based on our experience vetting the original protocol in multiple labs. The major change is an increase in input volume of biofluid from 200 µL to 500 µL. We found that the larger input volume led to more reproducible amounts of RNA isolation across multiple experiments. We have also included footnotes to explain changes we made to manufacturers' protocols, and observations that were made while carrying out the protocols.