With isobutyl chloroformate as active agent, Glycocholic acid-glycyl-1-13C, a 13C-breath test substrate and applications in clinical practice, was synthesized by one pot method of condensation of glycine-1-13C directly with cholic acid. 13C labelled Glycocholic acid was characterized by HPLC, LC-MS and 1HNMR. The yield of 13C labelled glycocholic acid was 80.3% (based on glycine-1-13C), with 99.1% atom13C abundance, 99.2% optical purity and 97.7% chemical purity, at optimized conditions, which were n(isobutyl chloroformate) : n(cholic acid) : n(glycine) : n(triethylamine)=1 : 1 : 1 : 1, 10 oC reaction temperature and 1 h reaction time.