Disulfide bridges are essential for maintaining the structure and function of proteins. Traditionally, studies of the disulfide bonds require expensive equipment and high purity of the protein sample, therefore, the development of simpler techniques is warranted. Here, were present a novel protocol for the detection of disulfide bonds in proteins, which is based on the labeling reduced disulfide bridges with a high molecular weight (HMW) maleimide derivative. After irreversible blocking of free thiol groups of proteins, the labeling of new thiols released from disulfide bridges with a high-molecular-weight (HMW) maleimide derivative is performed. To confirm localization of cysteines involved in the formation of disulfide bonds, cysteine mutagenesis was conducted. For validation, aquaporin 5 (AQP5) and transient receptor potential cation channel subfamily V member 4 (TRPV4) proteins were tagged with FLAG (DYKDDDDK) on N-termini. Increase in MW of the target proteins from immunoblot indicated the presence of disulfide bonds. No bands with increased MW were detected in AQP5, while TPRV4 cysteines at disulfide bridges-constituting positions 639, 645, 652, 660, 770 were detected and confirmed by cysteine mutagenesis. These data indicate that the proposed technique is feasible and effective for the detection of protein disulfide bonds.