RNA processing by the splicing machinery removes intronic sequences from pre-mRNA to generate mature mRNA transcripts. Many splicing events occur co-transcriptionally when the pre-mRNA is still associated with the transcription machinery. This mechanism raises questions regarding the number of spliceosomes associated with the pre-mRNA at a given time. In this protocol, we present a quantitative FISH approach that measures the ratio of intensities between two different spliceosomal components associated on a nascent mRNA, and compares to the number of introns in the mRNA, thereby calculating the number of spliceosome complexes assembled with each transcript.