NHS esters are commonly used reactive groups in crosslinking chemistry. We first demonstrated that NHS esters can react with the side-chain thiol group of cysteine and observed lysine-cysteine (K-C) crosslinks when using homobifunctional crosslinkers. However, in conventional crosslinking workflows, K-C crosslinks are difficult to identify. To address this, we explored factors affecting the stability of K-C crosslinks within the experimental pipeline. By employing an optimized workflow, we were able to consistently identify a greater number of K-C crosslinking sites while maintaining the detection of lysine-lysine (K-K) crosslinks.