Regioselective acylation of glycoconjugates with malonic acid was achieved by using phenyl β-d-glucopyranoside as a model glycoside, malonic acid, and tert-butyl isocyanide in aprotic solvents. Structural elucidation of phenyl 6‘-O-malonyl-β-d-glucopyranoside was performed by 1H and 13C NMR spectroscopy and high-performance liquid chromatography−atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC−APCI-MS/MS). This one-step reaction opens the way to the preparation of reference substances which are required for the spectroscopic identification of malonylated glycosides in complex natural matrices. Keywords: Phenyl 6‘-O-malonyl-β-d-glucopyranoside; malonylated glycosides; HPLC−MS/MS; atmospheric pressure chemical ionization (APCI)