The discovery of an acid labile metabolite of olsalazine, tentatively identified as olsalazine-O-sulphate, is described. Due to its acid labile properties, this conjugate was previously co-determined with olsalazine. Based on this finding, a new assay has been developed making it possible to determine both olsalazine and olsalazine-O-sulphate simultaneously. The initial aim of this study was to simplify an original analytical method in order to improve sample throughput without loss of sensitivity. However, during this work, a previously unidentified acid labile metabolite was discovered. The characterization of this metabolite is described here.