This study proposes a new method for the simultaneous determination of free glycerin, mono glycerides, diglycerides and triglycerides in biodiesel by GC/FID using the derivating agent N,O-bis (trimethylsilyl)trifluoracetamide (BSTFA) and the only internal standard (n-tetradecane). The silylation reaction was optimized. The sensitivity, precision and repeatability of method were evaluated.