A competitive enzyme-linked immunosorbent assay (ELISA), based on polyclonal antibodies, was developed to measure didecyldimethylammonium chloride (DDAC). The anti-DDAC polyclonal antibodies recognized the free DDAC in an indirect competitive ELISA. With this assay, the detection limit was 8 μg/mL, and 50% inhibition was reached at 29 μg/mL. The antibodies recognized the aliphatic chain of the DDAC molecule. They also cross-reacted with compounds having 10-12-carbon aliphatic chains, such as fatty acids, fatty alcohols, or n-decyl α-D-glucoside. The competitive ELISA was used to measure the amount of DDAC present in several available antisapstain products. For example, with F2A (15% DDAC), a linear relationship between the ELISA results and the theoretical values was observed in a range between 20 and 50 ppm DDAC. The measured concentrations of DDAC in other commercial products, F2, NP-1, and Bardac 2280, were in close agreement with the values reported by the manufacturer.