Abstract Summary Mass spectrometry imaging (MSI) can reveal biochemical information directly from a tissue section. MSI generates a large quantity of complex spectral data which is still challenging to translate into relevant biochemical information. Here, we present rMSIproc, an open-source R package that implements a full data processing workflow for MSI experiments performed using TOF or FT-based mass spectrometers. The package provides a novel strategy for spectral alignment and recalibration, which allows to process multiple datasets simultaneously. This enables to perform a confident statistical analysis with multiple datasets from one or several experiments. rMSIproc is designed to work with files larger than the computer memory capacity and the algorithms are implemented using a multi-threading strategy. rMSIproc is a powerful tool able to take full advantage of modern computer systems to completely develop the whole MSI potential. Availability and implementation rMSIproc is freely available at https://github.com/prafols/rMSIproc. Contact pere.rafols@urv.cat Supplementary information Supplementary data are available at Bioinformatics online.