Summary: A variety of crosslinking reagents with a maleimide group are widely used for crosslinking proteins to proteins, or proteins to other biomolecules. A key challenge in maleimide crosslinking technology has been the development of reliable and reproducible methods for quantitation the amount of maleimide linked to a single protein or antibody. Maleimides can be directly assayed spectrophotometrically at 302 nm. However, the small extinction coefficient of 620 M-1cm-1 renders this assay insensitive, and the assay is further complicated by the protein absorbance at the same wavelength. There are a few methods available for quantitating the number of maleimide groups by indirect ways. And in many cases, the maleimide ratio is only estimated by experience.