Fluorescent substrates for beta-lactamases having the general formula shown above are provided. Z includes a fluorophore or chromophore and includes a group that may link to the lactam-containing group; R.sub.1 and R.sub.2 are independently selected from H, aliphatic, aromatic, alkyl, and acyl; R.sub.4 may be H and lower alkyl; B may be H, physiologically acceptable salts or metal, ester groups, ammonium cations, --CHR.sub.5OCO(CH.sub.2).sub.nCH.sub.3, --CHR.sub.5OCOC(CH.sub.3).sub.3, acylthiomethyl, acyloxy-alpha-benz, deltabutyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, beta-morpholinoethyl, dialkylaminoethyl, and dialkylaminocarbonyloxymethyl, in which R.sub.5 is selected from the group consisting of H and lower alkyl; n is an integer between 0 and 10, inclusive; and A may be S, O, SO, SO.sub.2 or CH.sub.2. In embodiments, the beta-lactam ring may be cleaved by a beta-lactamase enzyme effective to free a fluorophore. Methods of assaying beta-lactamase activity and monitoring expression in systems using beta-lactamase as a reporter gene are also disclosed. ##STR00001##