Abstract: The invention relates to a competitive binding assay technique for substances (e.g. small molecules having no antigenic determinant) which can serve as substrates for coenzyme-requiring enzymes. The technique involves contacting a liquid sample with an assay system including (i) a substrate analogue and (ii) a moiety capable of binding specifically to the substrate without catalyzing the conversion of substrate to product. The substrate analogue is a substance capable of binding to said moiety competitively with any of the substrate present in the sample. The presence or amount of the substrate is determined by monitoring the extent to which there are formed or remain complexes between the moiety and substrate or substrate analogue.