The measurement of gluconate is required in the framework of nuclear waste management. Liquid chromatography coupled with amperometric detection proved to be insufficiently selective to quantify gluconate in radioactive effluents due to the complexity of the matrix. An analytical method based on a desalting step and quantification with liquid chromatography coupled to mass spectrometry was therefore developed. As gluconate may be present as several chelates, speciation simulation studies enabled to determine the prevailing chelate form depending on experimental conditions and thus to optimize sample preparation. The analytical method was successfully applied to real nuclear effluents, with a limit of quantification of 2.6 × 10−5 mol L−1.