Abstract Gene perturbation is essential for uncovering gene function and its involvement in various biological processes and diseases. GPSAdb 2.0 (https://www.gpsadb.com/) presents a significantly expanded and improved platform for exploring gene expression changes following gene knockdown across diverse cell lines. Compared to GPSAdb 1.0, this new version offers several key upgrades: (i) a substantial increase in data coverage, now featuring 7665 gene knockdown groups, 42 235 samples, and 2810 perturbation genes across a broader array of cell lines and biological conditions; (ii) the introduction of new tools, including “BioTrigger” for flexible gene set enrichment analysis and “fastGPSA” for rapid, advanced differential expression analysis. With these enhancements, GPSAdb 2.0 provides researchers with a powerful resource for investigating gene perturbation effects and gaining deeper insights into transcriptional regulation, making it an invaluable resource for studies on gene regulation, disease mechanisms, and potential therapeutic targets.