The invention discloses an Fmoc-protected 
amino acid purity and related substance 
analysis method. The method comprises the following steps of S1, preparing an Fmoc-Osu 
impurity control solution, andenabling the ratio of Fmoc-Osu to Fmoc-protected 
amino acid to be (0.1-1.0): 100 to obtain an Fmoc-Osu 
impurity control solution; S2, preparing a related 
impurity control solution, enabling the ratioof single related impurity to Fmoc-protected amino acids to be 0.1-1.0: 1 100, obtaining a plurality of reference substance solutions, etc. According to the invention, a mobile phase, an impurity control solution and the 
gradient elution conditions are comprehensively designed and optimized, the 
analysis method is applied to the operation process of the 
system, the baseline is smooth, no interference peak basically exists in a blank test, the impurity separation effect is prominent, and the specificity is good. The 
test data shows that the 
detection limit can achieve 0.002%, the sensitivity isextremely high, the detection result can provide the reliable and effective information for the research, the production and the purification of the product, the 
solid foundation is established for the protected 
amino acid quality control standard, and the positive effect is provided for the 
quality control of the final product.