Platycosides, main pharmacological effective compounds, are known to have several biological activities, including anti-obesity, anti-cancer and anti-diabetes. Although enzymatic preparation of platycosides was considered as effective method to obtain them, few analytical methods have been reported on process control. In the present study, we developed an application of reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of six platycosides during the process of enzymatic preparation of deapio-platycodin D (dPD) and platycodin D (PD). The method employed a Hypersil ODS2 analytical column (250 × 4.6 mm I.D., 5 μm) coupled with UV detector at 210 nm with flow rate of 1.0 mg mL−1. A step gradient of acetonitrile–water (v/v) was applied, leading to a sample analysis of 60 min. The method was validated in terms of linearity, sensitivity, precision and accuracy. The correlation coefficients (R 2) for calibration curves of platycosides were in the range of 0.9995–1.0 when the linearity range was from 0.85 to 10.2 mg mL−1. The proposed RP-HPLC method was successfully applied to the analysis of enzymatic preparation study and the recoveries of platycosides were in the range of 96.22–102.56% with RSD <3.3%. The method could be of use for rapid and routine evaluation of the quantity of platycosides during the enzymatic preparation process.