Recently, new software tools have been developed for improved protein quantification using mass spectrometry (MS) data. However, there are still limitations especially in high-sample-throughput quantification methods, and most of these relate to extensive computational calculations. The mass accuracy precursor alignment (MAPA) strategy has been shown to be a robust method for relative protein quantification. Its major advantages are high resolution, sensitivity and sample throughput. Its accuracy is data dependent and thus best suited for precursor mass-to-charge precision of ∼1 p.p.m. This protocol describes how to use a software tool (ProtMAX) that allows for the automated alignment of precursors from up to several hundred MS runs within minutes without computational restrictions. It comprises features for 'ion intensity count' and 'target search' of a distinct set of peptides. This procedure also includes the recommended MS settings for complex quantitative MAPA analysis using ProtMAX (http://www.univie.ac.at/mosys/software.html).