Pre-mRNA splicing is an essential process for gene expression regulation in eukaryotes. It is a co-transcriptional step that removes introns and ligates exons into mature RNA molecules. Splicing is performed by a complex macromolecular machinery composed of snRNAs and more than a hundred proteins. Alteration of splice sites or spliceosome protein components leads to altered splicing efficiency and can be related to the development of several diseases. Recently, the use of small molecule splicing inhibitors has shown some processes can be stalled in vitro due to reduced speed of splicing reactions. Splicing dynamics and kinetics haves been extensively studied in vitro, providing an excellent understanding of the composition of spliceosome intermediates and splicing efficiency using different pre-mRNA substrates. However, analysis in cultured cells can accomplish variations in the splicing process, related, for example, to different genetic backgrounds or disease models. In this sense, this methods collection will search for approaches to analyze pre-mRNA splicing in cell cultures. These methods include but are not limited to: (1) methods that promote visualization of pre-mRNA splicing in live cells and (2) methods directed to analyze splicing efficiency and inhibition in cells, which also might have implications to cellular kinetic parameters.