This study investigates the quantification of thiamine (vitamin B1) and riboflavin (vitamin B2) in Pleurotusostreatus (oyster mushrooms) cultivated on various lignocellulosic substrates using a simplified specific High-Performance Liquid Chromatography (HPLC) method. The substrates included paddy, soya bean, and red gram. Mushroom samples were cultivated, harvested, dried, and ground before being analyzed. The HPLC system used Agilent TC-C18 columns with a mobile phase ratio of acetonitrile and water (90:10) and detection wavelengths of 245 nm for thiamine and 268 nm for riboflavin. Results indicated consistent retention times and accurate concentration measurements, with thiamine concentrations ranging from 3.003 µg/ml to 3.0229 µg/ml and riboflavin concentrations showing more variability from 7.22 µg/ml to 11.050 µg/ml. The method demonstrated reliability and precision, suggesting its suitability for analyzing these vitamins in similar sample matrices. Future research could explore the causes of variability in riboflavin concentrations and extend the method to other types of samples.