3’ end cleavage and polyadenylation are required steps in pre-mRNA maturation. The rate at which 3’ end cleavage occurs can determine the temporal availability of mRNA for subsequent function throughout the cell and is likely tightly regulated. While there are numerous high-throughput methods for global profiling of RNA maturation rates, the study of pre-mRNA 3’ end cleavage kinetics has remained limited to low-throughput approaches, and the temporal regulation of polyadenylation site choice that determines the composition of the 3’ UTRs of mRNAs remains poorly understood. This research project seeks to address this gap by introducing a novel genome-wide, site-specific methodology for estimating rates of pre-mRNA 3’ end cleavage, using metabolic labeling of nascent RNA, high-throughput sequencing, and mathematical modeling. Using in-silico simulations of nascent RNA-seq data, we show that our approach can accurately and precisely estimate cleavage half-lives for both constitutive and alternative sites. In Drosophila melanogaster S2 cells, we find that cleavage rates are fast but highly variable across sites, with alternative events being slowest. This variability in rates is underpinned by distinctive sequence elements, where an A-rich region upstream of the cleavage site, a U-rich element downstream of the cleavage site, and a higher density of polyadenylation signals, lead to faster cleavage reactions. Assessment of Polymerase II dynamics around cleavage sites reveals that cleavage rates are associated with the localization of RNA Polymerase II at the end of a gene and faster cleavage leads to quicker degradation of downstream read-through RNA. This approach for estimating pre-mRNA 3’ end cleavage kinetics opens new possibilities in the study of co-transcriptional regulation of mRNA expression and transcription termination across cellular states.