This paper describes the optimization of a method of simultaneous determination of glycerides, free and total glycerol in biodiesel ethyl esters from castor oil by using gas chromatography. Changes were proposed for the methods ASTM D 6584 and EN 14105 in order to determine these by-product contaminants in biodiesel from castor oil. The silylation reaction for this biodiesel was optimized, and 250 mu L MSTFA was used. Its accuracy values were between 70% and 120% with RSD <11%. The identification of monoricinolein and diricinolein was made by gas chromatography with mass spectrometry detection (GC-MS). The matrix effect (ME) was investigated and considered low for glycerol, mono-and diolein; it was medium for triolein. The method was robust even when there were variations in the matrix. It was also successfully used for the determination of glycerides, free and total glycerol in samples of biodiesel from castor oil. (C) 2011 Elsevier Ltd. All rights reserved.