A method for quantification of diarylheptanoids in Curcuma comosa rhizomes and selected pharmaceutical preparations was established by using HPLC-diode array detector (DAD). The chromatographic separation of three diarylheptanoids [(3 S )-1-(3,4-dihydroxy-phenyl)-7-phenyl-(6 E )-6-hepten-3-ol ( 1 ), (3 R )-1,7-diphenyl-(4 E ,6 E )-4,6-heptadien-3-ol ( 2 ), and (3 S )-1,7-diphenyl-(6 E )-6-hepten-3-ol ( 3 )] was performed on a Luna C₁₈ analytical column using gradient elution with 0.5% acetic acid in water and acetonitrile with a flow rate of 1 mL/min and a column temperature of 35°C. The calibration curves for the analytes showed good linearity ( R ²>0.999), high precision (relative standard deviation (RSD) <2%) and acceptable recovery (98.35–103.90%, RSD <2%). The limit of detection (LOD) and limit of quantification (LOQ) were 0.06–0.22 and 0.18–0.69 µg/mL, respectively. The results of all validated parameters were within the limits according to the International Conference on Harmonization (ICH) Guidelines. The established method was successfully applied for qualitative and quantitative determination of the three constituents in different samples of C. comosa and some commercial products in capsules. The simplicity, rapidity, and reliability of the method could be useful for the fingerprint analysis and standardization of diarylheptanoids, which are responsible for the estrogenic activity in raw materials and herbal medicinal products of C. comosa .