Provided are fluorescent substrates for .beta.-lactamases having the general formula I: ##STR00001## in which R is a benzyl, 2-thienylmethyl, or cyanomethyl group; R' is selected from the group consisting of H, physiologically acceptable salts or metal, ester groups, ammonium cations, --CHR.sub.2OCO(CH.sub.2).sub.nCH.sub.3, --CHR.sub.2OCOC(CH.sub.3).sub.3, acylthiomethyl, acyloxy-alpha-benzyl, deltabutyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, .beta.-morpholinoethyl, dialkylaminoethyl, and dialkylaminocarbonyloxymethyl, in which R.sub.2 is selected from the group consisting of H and lower alkyl; A is selected from the group consisting of S, O, SO, SO.sub.2 and CH.sub.2; and Z is a donor fluorescent moiety. Also provided are methods of use of the compound of general formula I.