Uptake of the fluorescent probe 1‐N‐phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram‐negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram‐negative bacteria.