The catalytic removal of introns via pre-mRNA splicing during pre-mRNA processing is an essential component of eukaryotic gene expression. Splicing is a pervasive and highly regulated process that is commonly mis-regulated in human disease. To better understand the mechanisms by which splicing is regulated and mis-regulated, genome-wide assays with increased resolution for splicing are required. To this end, I developed multiplexed primer extension sequencing (MPE-seq); a targeted RNA sequencing method that increases precision in genome-wide quantification of splicing isoforms and allows for the genome-wide quantification of transcripts based on which chemical step of splicing has occurred. Understanding how the spliceosome processes its composite of pre-mRNA substrates through the two chemical steps required for mature mRNA production will be essential to deciphering splicing regulation, and its mis-regulation in human disease. To address this, I have measured the genome-wide in vivo rates of each step of pre-mRNA splicing across the genome-wide complement of splicing substrates in budding yeast. This was achieved by coupling metabolic RNA labeling, MPE-seq, and first order kinetic modeling. I demonstrate that there exists a wide variety in rates by which different introns are removed, that splice site sequences are primary determinants of 1st step rates, and that the 2nd step is generally faster than the 1st. Additionally, I find that the ribosomal protein genes (RPGs) are spliced faster than non-RPGs (nRPGs) at each step and that RPGs share distinct and evolutionarily conserved cis-features that differentiate them from nRPGs and may contribute to their faster splicing. I was able to measure changes in splicing rate in a 1st step splicing mutant that reveal a significant impact on both 1st and 2nd step rates. Moreover, we find RPGs are significantly more slowed at each step compared to nRPGs, suggesting transcript specific impacts on splicing. Additionally, these data uncover a coupling between transcription and 1st and 2nd step splicing rates that suggests co-transcriptional splicing is an important determinant of splicing rates.