Percent Spliced-In (PSI) values are commonly used to report alternative pre-mRNA splicing (AS) changes. Previous PSI-detection tools were limited to specific AS events and were evaluated by in silico RNA-seq data. We developed PSI-Sigma, which uses a new PSI index, and we employed actual (non-simulated) RNA-seq data from spliced synthetic genes (RNA Sequins) to benchmark its performance (i.e., precision, recall, false positive rate, and correlation) in comparison with three leading tools (rMATS, SUPPA2, and Whippet).