Motivation: Understanding the occurrence and regulation of alternative splicing (AS) is a key task towards explaining the regulatory processes that help to shape the complex transcriptomes of higher eukaryotes. With the advent of high-throughput sequencing of RNA, the diversity of AS transcripts could be measured at an unprecedented depth. Although the catalog of known AS events has ever grown since, novel isoforms are commonly observed when working with less annotated organisms, in the context of diseases, or within large populations. Whereas an identification of complete isoforms is technically challenging or expensive, focussing on single splicing events as a proxy for transcriptome characteristics is fruitful for differential analyses. Results: We present SplAdder, an alternative splicing toolbox, that takes RNA-Seq alignments and an annotation file as input to i) augment the annotation based on RNA-Seq evidence, ii) identify alternative splicing events present in the augmented annotation graph, iii) quantify and confirm these events based on the RNA-Seq data, and iv) test for significant quantitative differences. Availability: Source code and documentation are available for download at github.com/ratschlab/spladder. Example data, introductory information and a small tutorial are accessible at bioweb.me/spladder.