The thin-layer chromatography (TLC) combined with high-performance liquid chromatography (HPLC) has been proved to be a quick and valid method to detect the intermediates and the end-product created during the chemosynthesis process of vinorelbine (VB). This paper gives a detailed investigation on the results of two determination methods when the condition of the detection changed. It shows that when TLC developer is consisted of petroleum ether, chloroform, acetone, and diethyl amine (23.5:12:2:2.5, v/v/v/v), vinblastine sulfate (VBS), anhydrovinblastine (AHVB), and VB can be separated specifically. When the mobile phase of HPLC is a mixture of methyl alcohol, acetonitrile, diethyl amine, and high purity water (420:252:3:225; v/v/v/v), adjusted with orthophosphoric acid to pH 6.5, the intermediates and the resultants of the chemosynthesis of VB can be determined effectively. It can also be used to fix quantify of the resultants. The calibration curve for VB shows good linearity in the two mass concentration ranges of 0.0100-0.0500 mg/mL (r = 0.9956) and 0.00600-0.0100 mg/mL (r = 0.9978), respectively. The limit of detection of HPLC for VB is 0.200 microg/mL.