Pre-mRNA splicing is a highly conserved step in regulating eukaryotic gene expression levels and transcript identities. Non-coding intronic sequences must be precisely removed from nascent transcripts through 2 sequential transesterification reactions. In every splicing event, a large ribonucleoprotein complex named the spliceosome must assemble anew and catalyze the reactions through a chain of intermediate steps. The efficiency of each of these steps is transcript-specific and regulates gene expression. However, what cis-elements determine these efficiencies is not well understood. Our lab has previously developed MPE-seq, a targeted RNA-seq method that allows the simultaneous detection of rare splicing isoforms from all introns in the genome. Coupling this method with metabolic labeling enables the quantification of splicing efficiency at each of the two transesterification steps. In this thesis, I demonstrate my work to further develop this method to allow large-scale comparative studies on strains carrying different variations of the same gene. With this method, I worked towards setting up a system using two distinct classes of transcripts in Saccharomyces cerevisiae to identify and understand the cis-elements that lead to their different splicing regulations.