Cross-linking mass spectrometry (XL-MS) is capable of identifying protein-protein interactions (PPIs) in complex protein mixtures and providing valuable structural information of proteins in their native states. XL-MS is performed by using specifically designed cross-linkers that connect two amino acids in close proximity within the same or different proteins with a covalent bond. Upon enzymatic digestion, this generates cross-linked peptides, which are identifiable by liquid chromatography tandem mass spectrometry, (LC-MS/MS). Despite many recent outstanding studies employing XL-MS approaches, there are still several limitations that have to be overcome in order to establish XL-MS as a robust workflow that can be used in service infrastructures. Detecting and identifying cross-linked peptides is one of the challenging steps due to the broad dynamic range and high complexity of proteomes, which often interfere with the identification of low-abundance cross-linked peptides in complex mixtures. Therefore, careful consideration is very important when selecting a peptide separation approach in order to enhance sample recovery and cross-link identifications. This work aims to optimize four fractionation methods, Size Exclusion Chromatography (SEC), High-pH Reversed-Phase Chromatography, Porous Graphitic Carbon (PGC)-HPLC, and PGC stage tips, that are available at our facility. The gradient LC-MS/MS profiles and MS acquisition parameters are first optimized and then applied to validate these fractionation methods for separating cross-linked peptides from high-complexity samples (E. coli cell lysates) prior to MS analysis. In combination with the [MR1] enrichable cross-linker PhoX, approximately 2,000-5,800 unique cross-linked peptides and 100-700 protein-protein interactions (PPIs) from E. coli lysates are identified across the methods. From the comparison of four different fractionation approaches, SEC and PGC-HPLC methods are able to identify more crosslinks than other methods. Altogether, the validated fractionation method developed here will be useful as a guideline for the XL-MS field and can be applicable for several types of cross-linkers and other proteome-wide XL-MS studies. This work will lead to a robust and widely applicable XL-MS protocol in the EMBL proteomics core facility and EMBL researchers and researchers across the EMBL member states.