Iodoacetamide is widely used as an alkylating agent in biochemical and molecular biology research. It is primarily applied to modify cysteine residues in proteins, preventing disulfide bond formation and protecting thiol groups from oxidation during protein sequencing or electrophoresis. This modification enhances the stability and accuracy of protein analysis in techniques like SDS-PAGE and mass spectrometry.

It is also employed in proteomics workflows to irreversibly inhibit cysteine proteases, helping to control enzymatic activity in sample preparation. Due to its ability to cross cell membranes, iodoacetamide can be used in cellular studies to probe redox environments or disrupt thiol-dependent processes.

In addition, it serves as an inhibitor of certain metabolic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), making it useful in studies of cellular metabolism and enzyme function. However, because of its reactivity and potential toxicity, handling requires care, especially in live-cell applications.