The objective of this study was to establish improved methods for determination of the activities of the key phenolic biosynthesis enzymes. The pericarp of litchi (Litchi chinensis), an important evergreen fruit crop cultivated in south China, was used as material. Two-point method and enzymatic kinetic method combined with the use of photospectrometry and high performance liquid chromatography to analyze the formation of end products were applied. The key phenolic biosynthesis enzymes included phenylalanine ammonia-lyase (PAL), chalcone flavanone isomerase (CHI), dihydroflavonol 4-reductase (DFR) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT). The enzyme purification from crude extracts, enzyme activity protection and methods to dissolve substrates were all critical for reliable enzyme activity determinations. The possible problems during the activity determination of these enzymes and corresponding solutions were mentioned. The changes in PAL, CHI, DFR and UFGT activities measured by the improved methods in the pericarp of litchi during fruit development were generally consistent with previous reports. The improved methods are reliable and will provide important reference for phenolic biosynthesis studies.