Alternative Splicing (AS) plays a critical role in conferring phenotypic diversity, and has the potential to contribute to environmental acclimation/adaptation in plants. Current efforts to quantify AS in short-read RNA-seq samples either attempt to estimate transcript isoform abundance, or compare occurrences of various mutually exclusive splicing events. In this work we describe the conceptual development of Splice-site Strength, an explicit quantification of splice site utilisation at the level of individual base pairs; and describe its transcriptome-wide implementation in the bioinformatic tool SpliSER (Splice-site Strength Estimate from RNA-seq). We further demonstrate multiple applications of SpliSER, which have major implications for our understanding of AS.