Plant sterols are belong to triterpenes family of natural products which includes more than 200 different types of plant sterols and more than 4000 other types of triterpenes. The optimization of method, specially the derivatization step as well as the corresponding analytical validation, is the main goal of this study. The optimum temperature, time and reagent volume of derivatization step were obtained at 60°C, 60 minutes and 50 µL, respectively. A rapid and sensitive gas chromatographic–mass spectrometric method was developed and validated for quantitative analysis of the most common plant sterols (β-sitosterol, campesterol and stigmasterol) in 20 Turkish bread wheat cultivars using GC-MS-SIM. Separation of β-cholestanol (I.S), campesterol, stigmasterol and β-sitosterol was achieved on Rxi (5Sil MS) column (60 m×0.25 mm). The limits of detection for β-sitosterol, campesterol and stigmasterol were 0.074, 0.054 and 0.064 mg kg-1 , respectively with RSD ≤ 0.66%. The obtained concentrations of campesterol, stigmasterol and β-sitosterol from 20 Turkish bread wheat cultivars ranged from: 15.30 to 76.02, 4.27 to 23.23 and 303.21 to 682.66 mg kg-1 , respectively.