Substrates for .beta.-lactamase of the general formula I ##STR00001## in which one of X and Y is a fluorescent donor moiety and the other is a quencher (which may or may not re-emit); R' is selected from the group consisting of H, lower (i.e., alkyl of 1 to about 5 carbon atoms) and (CH.sub.2).sub.nOH, in which n is 0 or an integer from 1 to 5; R'' is selected from the group consisting of H, physiologically acceptable metal and ammonium cations, --CHR.sup.2OCO(CH.sub.2).sub.nCH.sub.3, --CHR.sup.2OCOC(CH.sub.3).sub.3, acylthiomethyl, acyloxy-alpha-benzyl, delta-butyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, beta-morpholinoethyl, dialkylaminoethyl, acyloxyalkyl, dialkylaminocarbonyloxymethyl and aliphatic, in which R.sup.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' and Z'' are linkers for the fluorescent donor and quencher moieties. Methods of assaying .beta.-lactamase activity and monitoring expression in systems using .beta.-lactamase as a reporter gene also are disclosed.