ObjectiveProtein microarrays provide a high-throughput platform to measure protein interactions and associated functions, and can aid in the discovery of cancer biomarkers. The resulting protein microarray data can however be subject to systematic bias and noise, thus requiring a robust data processing, normalization and analysis pipeline to ensure high quality and robust results. To date, a comprehensive data processing pipeline is yet to be developed. Furthermore, a lack of analysis consistency is evident amongst different research groups, thereby impeding collaborative data consolidation and comparison. Thus, we sought to develop an accessible data processing tool using methods that are generalizable to the protein microarray field and which can be adapted to individual array layouts with minimal software engineering expertise.ResultsWe developed an improved version of a previously developed pipeline of protein microarray data processing and implemented it as an open source software tool, with particular focus on widening its use and applicability. The Protein Microarray Analyser software presented here includes the following tools: (1) neighbourhood background correction, (2) net intensity correction, (3) user-defined noise threshold, (4) user-defined CV threshold amongst replicates and (5) assay controls, (6) composite ‘pin-to-pin’ normalization amongst sub-arrays, and (7) ‘array-to-array’ normalization amongst whole arrays.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3266-0) contains supplementary material, which is available to authorized users.