Abstract Summary Large-scale peptide mass spectrometry (MS)/MS reference libraries are essential for the comprehensive analysis of data-independent acquisition (DIA) MS datasets, providing a comprehensive set of spectra for identification and quantification of proteins. We have developed a novel web-based R-package (iSwathX) for combining reference libraries that is compatible with different DIA analysis software. This open-source web GUI automates the process of normalization and combination of spectral libraries and provides a user-friendly method for performing library format conversions, analysis and visualizations, with no need for programing familiarity. Availability and implementation iSwathX is freely accessible at https://biolinfo.shinyapps.io/iSwathX with the R-package and Shiny source code available from GitHub (https://github.com/znoor/iSwathX). Supplementary information Supplementary data are available at Bioinformatics online.