Background: Clausena harmandiana Pierre. (CH) contains various bioactive analytes with pharmacological benefits. Most researches were focused on carbazole analytes determined by isocratic high-performance liquid chromatography (HPLC), only few were focused on coumarin analytes and harvested location. Objective: To develop and validate gradient HPLC method to analyze the variance of nine target analytes contained in roots of CH grown naturally in four different provinces of Thailand. Materials and Methods: The analytical method was undertaken by gradient HPLC with 3% tetrahydrofuran in acetonitrile, and 0.05% phosphoric acid in water as mobile phases, on Hypersil ODS column (4.0 × 250 mm, 5 μm), at flow rate 1.0 mL/min and detected at wavelength 280 nm. The method was validated for system linearity, limit of detection, limit of quantitation, precision and accuracy. Results: The new-developed method was able to detect the nine target analytes in CH root. The validation showed the reliability of the method. All system suitability parameters were within the satisfied limits. The linear responses of method were observed at r2 ≥ 0.999 for all analytes. The obtained amount of nine analytes showed the biodiversity of contents in different provinces. Of the nine target analytes, the level of nordentatin and dentatin in coumarin groups were considerably high in plants collected from one specific province of Thailand. Conclusion: This study has shown that the new-developed method is reliable, precise, accurate and sensitive to determine and quantify the nine target analytes in CH. Nordentatin and dentatin obviously show the higher level in one specific province of Thailand.