A chiral chromatographic method for the determination of calcium L-5-methyltetrahydrofolate (L-5-MTHF-Ca) was established， and the feasibility of the method was evaluated by methodology validation. Methods high performance liquid chromatography is used. The chromatographic column is CHIRALPAK HSA chiral column (4 mm×150 mm， 5 μm). The mobile phase is phosphate buffer solution ∶ acetonitrile (90 ∶ 10， V ∶ V). The flow rate is 0.6 mL/min. The injection volume is 5 μL. The column temperature is 30 ℃ and the detection wavelength is 280 nm. The results shows that the method could separate L-5-MTHF-Ca well， the resolution is 3.24， and the linear relationship is good in the concentration range of 0.016~0.100 mg/mL， the correlation coefficient is 0.9997， the detection limit and quantitation limit are 3.11 μg/mL and 4.98 μg/mL respectively， the precision is 0.38%， and the average recovery is 103.71%. System suitability test results are good， but L-5-MTHF-Ca will slowly degrade over time， so sample analysis as soon as possible. The method is simple， accurate and can be used for the determination of L-5-MTHF-Ca in various foods and health products.