A high-performance liquid chromatographic (HPLC) reliable and sensitive method with diode array detection (DAD) system has been developed for the determination of secoisolariciresinol diglucoside (SDG) in flaxseed oil. An analytical methodology based on the sample extraction with methanol/water (80:20, v/v), subsequent purification of the sample and analysis of the extract by HPLC/DAD is proposed for the determination of SDG in flaxseed oil. The coefficient of determination of the standard calibration curve was 0.999, the limit of detection was 0.08μg/mL, and the limit of quantification was 0.27μg/mL. The recovery test was conducted adding four different concentrations of standard solution to the blank sample. The recovery ranged from 90% to 95%. To our knowledge, the presence and quantification of SDG in flaxseed oil has never been reported. The proposed method was tested to study the variation in SDG content in flaxseed oil during a shelf-life test to verify its applicability in quality control for oil industries. The dark and the low temperature of storing together allowed to preserve SDG. A slight pro-oxidant effect was observed for the addition of antioxidant to the flaxseed oil.