The ceramide (Cer) content of skin and glucosylceramide (GlcCer) intake affect skin moisture conditions, but their mutual relation in skin remains unclear. For clarification of that mutual relation, carbon stable isotopes (¹²C and ¹³C) are useful as a tracer. However, carbon isotopic measurement has not been applied to the study of clarifying their skin moisturizing effects. Therefore, we used gas chromatography / combustion / isotope ratio mass spectrometry (GC-C-IRMS) to ascertain the appropriate conditions for carbon isotopic measurements using synthesized Cer (SCer) in substitution for very low concentrations of Cer in skin. SCer was derivatized to trimethylsilylated SCer (TMS-SCer) quantitatively using N-trimethylsilylimidazole (TMSI) depending on the amount of SCer. The derivatization rates were 75–85%. Excess TMSI was removed using three cycles of hexane–water distribution. Under these conditions, carbon isotopic measurements of TMS-SCer conducted using GC-C-IRMS showed high repeatability and good inter-day variation (S.D. < 0.3‰). The carbon stable isotope ratio value (δ¹³C) of SCer calculated using a mass balance equation was compared with δ¹³C of underivatized SCer, which was regarded as the actual δ¹³C of SCer obtained using sealed tube combustion method. The difference between the calculated δ¹³C of SCer and δ¹³C of the underivatized SCer depended on the TMSI reagent supplier and on the number of hydroxyl groups to be derivatized in SCer. For accurate δ¹³C of Cer in skin using GC-C-IRMS, the measured δ¹³C of a target TMS-Cer must be calculated using a correction factor representing the difference in δ¹³C of underivatized standard SCer from that of TMS-standard SCer having a structure resembling that of the target Cer in skin. In addition, we show that the same lot of TMSI reagent from a specific supplier must be used throughout the experiments.