Manipulating gene expression is crucial for understanding gene function, with high-throughput sequencing techniques such as RNA-seq elucidating the downstream mechanisms involved. However, the lack of a standardized metadata format for small-scale perturbation expression datasets in public repositories hinders their reuse. To address this issue, we developed PerturbAtlas, an add-value resource that re-analyzes publicly archived RNA-seq libraries to provide quantitative data on gene expression, transcript profiles, and alternative splicing events following genetic perturbation. PerturbAtlas assists users in identifying trends at the gene and isoform levels in perturbation assays by re-analyzing a curated set of 122 801 RNA-seq libraries across 13 species. This resource is freely available at https://perturbatlas.kratoss.site as both raw data tables and an interactive browser, allowing searches by species, tissue or genomic features. The results provide detailed information on alterations following perturbations, accessible through both forward and reverse approaches, thereby enabling the exploration of perturbation consequences and the identification of potential causal perturbations.© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.