Preparation of a seed extraction by pre-treatment is an essential step in the extraction of thymoquinone by preparatory HPLC. A total of 3 different methods were used for the preparation of N. sativa seed extracts with various solvents. It was understood that using methanol, Hexane and aqueous extract as solvents for the process of seed extract had the highest yield of thymoquinone respectively, compared to other solvents like ethanol and benzene. Pre-treatment of N. sativa using methanol as a solvent