Abstract: Nowadays, ¹³C-fatty acid breath test has been widely used in detection of gastric emptying. A general analytical method of the isotope abundance and chemical purity of ¹³C-fatty acids was developed with gas chromatography-mass spectrometry (GC/MS), based on the fatty acids with γH shared Mclaffery rearrangement ion. Stable isotope labeled ¹³C-octanoic acid and ¹³C-acetic acid developed by Shanghai Research Institute of Chemical Industry are determined by the new method, which isotope abundances are 98.7% and 98.5%, respectively. The precision expressed in RSD is less than 0.06%. Meanwhile, “isotope memory effect” of GC/MS for detecting samples was investigated. Then the effect of sample concentration for isotope abundance detection was also studied. Isotope abundance results of ¹³C-octanoic acid determinate by EI source and ESI source, respectively, are basically same, both of them have good precision. The isotope abundance and chemical purity can be determined by the developed method simultaneously.