Crosslinking mass spectrometry (XL-MS) has received considerable interest due to its potential to investigate protein-protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison to other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by use of bi-functional crosslinking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by mass spectrometry. XL-MS has received the required boost to tackle more complex samples with recent advances in crosslinking chemistry with MS-cleavable or reporter-based crosslinkers and faster, more sensitive and more versatile mass spectrometry platforms. This protocol provides a detailed description of our optimized conditions for a full proteome native protein preparation followed by crosslinking using the gas-phase cleavable crosslinking reagent DSSO. Following crosslinking, we demonstrate extensive sample fractionation and significantly simplified data analysis with XlinkX in Proteome Discoverer and subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within approximately 10 d including structural investigations.