Abstract: Objective:To compare three methods for the determination of L-threonine. Methods:Three L-threonine determination methods:paper chromatography, thin layer chromatography, high performance liquid phase PITC pre-column derivatization were compared and optimized. And the above three methods were used to determine the L-threonine content and spike recovery in 10 fermentation broth samples. Results:The determination coefficient of paper chromatography, thin layer chromatography and high performance liquid phase PITC pre-column derivatization were 0.9938, 0.9971 and 0.9992, and the detection limits were 5, 7 and 1.2 μg/L.In terms of the variance of each group of samples, the difference of the high performance liquid phase PITC pre-column derivatization was the smallest, and the paper chromatography was similar to the thin-layer chromatography. The average recoveries of the three methods were 95.60%, 95.10%, and 99.30%. Conclusion:The three methods are all effective for detecting the content of L-threonine in samples. In terms of accuracy, high performance liquid phase PITC pre-column derivatization has the best accuracy, followed by paper chromatography and thin layer chromatography. In summary, paper chromatography and thin-layer chromatography are low-cost and suitable for content determinations that do not require high accuracy. High performance liquid phase PITC pre-column derivatization is accurate, high cost, fast and suitable for accurate determination.