Abstract Background Protein microarray is a well-established approach for characterizing activity levels of thousands of proteins in a parallel manner. Analysis of protein microarray data is complex and time-consuming, while existing solutions are either outdated or challenging to use without programming skills. The typical data analysis pipeline consists of a data preprocessing step, followed by differential expression analysis, which is then put into context via functional enrichment. Normally, biologists would need to assemble their own workflow by combining a set of unrelated tools to analyze experimental data. Provided that most of these tools are developed independently by various bioinformatics groups, making them work together could be a real challenge. Results Here we present PAWER, the online web tool dedicated solely to protein microarray analysis. PAWER enables biologists to carry out all the necessary analysis steps in one go. PAWER provides access to state-of-the-art computational methods through the user-friendly interface, resulting in publication-ready illustrations. We also provide an R package for more advanced use cases, such as bespoke analysis workflows. Conclusions PAWER is freely available at https://biit.cs.ut.ee/pawer.