Disclosed are nitro-substituted squaraine reporter dyes and methods using such dyes for detecting nitroreductase enzyme activity and nitroreductase gene expression in cellular assays. The dyes are of the structure: in which Z1 and Z2 independently represent a phenyl or a naphthyl ring system; X and Y are selected from oxygen, sulphur, —CH═CH— and the group: R1 and R2 are selected from C1-C4 alkyl, —(CH2)n—P, —{(CH2)2—O}p—R6 and group W; where P is selected from COOR7, SO3− and OH, W is mono- or di-substituted nitrobenzyl, R6 is methyl or ethyl, R7 is selected from H, C1-C4 alkyl and CH2OC(O)R8, where R8 is methyl, or t-butyl, n is an integer from 1 to 10, and p is an integer from 1 to 3; R3 and R4 are selected from hydrogen, NO2, halogen, SO3−, C1-C4 alkoxy and —(CH2)m—COOR7; where R7 is hereinbefore defined and m is 0 or an integer from 1 to 5; R5 is C1-C6 alkyl optionally substituted with COOR7, SO3−, or OH; where R7 is hereinbefore defined; and at least one of groups R1, R2, R3 and R4 comprises at least one NO2 group. Also provided are methods for screening for a test agent whose effect upon nitroreductase enzyme activity and nitroreductase gene expression is to be determined.