To establish a GC-MS method for the determination of calophyllolide in Calophyllum inophyllum L. and its application in quality control of natural medicine. Methanolic extract from Calophyllum inophyllum L. was prepared. Calophyllolide was isolated, purified and then identified to serve as a chemical marker for development and validation a GC-MS method according to ICH guideline and USP. The established method was proved to be useful in quality control with the aid of Principle Component Analysis (PCA), a multivariate data analysis technique. The proposed method was selective against mass spectral deconvoluting software AMDIS32. The linear range was from 3.125–50 μg·mL −1 . The RSD of intra-and inter-day precision variations were less than 3% and the mean recovery was (101.22 ± 1.98) % (RSD = 1.95%). PCA was used to indicate a differentiation of batches in production as well as proving that calophyllolide has influence on the discrimination of extracts from different batches. The proposed capillary gas chromatography/mass spectrometric method for the determination of calophyllolide obtained from resin of Calophyllum inophyllum L. nut oil showed its suitability, simplicity, rapidity and precision, and it can be used for chemical assay. This is the first time such a technique was reported in literature. The multivariate data analysis technique was employed to provide an extensive but inclusive method for quality control of the extracts, to ensure consistency for batch to batch of extracts to be used for product development.