Within the research field of cross-linking mass spectrometry (XL-MS), the most commonly used cross-linking reagents are succinimide-ester-based (e.g., disuccinimidyl suberate (DSS)). These reagents primarily cross-link lysine side chains. So far, they have predominantly been used to investigate protein structures at neutral to slightly basic pH (7.0–8.5) to ensure the reactivity of the primary amine of the lysine side chain. However, disease-related molecular processes are not limited to such pH ranges; e.g., some important biological pathways are active in acidic intracellular compartments. The applicability of lysine-reactive cross-linking reagents to low-pH conditions remains unclear. Here, we cross-linked a mixture of eight model proteins at eight different pH conditions (pH 4.0–7.5) to investigate the pH dependency of DSS. DSS was able to cross-link proteins even at pH 4.0, but a clear decrease in the cross-linking efficiency was observed when the pH was lowered. Nevertheless, at pH 5.0, approximately half of the number of cross-links observed at pH 7.5 could still be identified. These findings highlight the ability of succinimide-based cross-linking reagents to be useful in probing the structure of proteins in a slightly acidic environment.