Nuclear run-on (NRO) is a method that measures transcriptional activity via the quantification of biochemically labeled nascent RNA molecules derived from nuclear isolates. Widespread use of this technique has been limited because of its technical difficulty relative to steady-state total mRNA analyses. Here we describe a detailed protocol for the quantification of transcriptional activity in human cell cultures. Nuclei are first isolated and NRO transcription is performed in the presence of bromouridine. Labeled nascent transcripts are purified by immunoprecipitation, and transcript levels are determined by reverse-transcription quantitative PCR (RT-qPCR). Data are then analyzed using standard techniques described elsewhere. This method is rapid (the protocol can be completed in 2 d) and cost-effective, exhibits negligible detection of background noise from unlabeled transcripts, requires no radioactive materials and can be performed from as few as 500,000 nuclei. It also takes advantage of the high sensitivity, specificity and dynamic range of RT-qPCR.