A procedure has been developed for the determination of penicilloates based on the reduction of Cu^II and detection of Cu^I using neocuproine. The sensitivity range is 5.6–56 µM penicilloate and the standard deviation was found to be between 1 and 2%. All the penicilloates tested reduced Cu^II under the conditions of the assay and a slight modification allowed a reasonably accurate determination of the level of this biologically inactive ingredient in a mixture with the parent penicillin. The deterioration of solutions of benzylpenicillin was also studied.