A reliable method with ensured traceability of the measurement results for free and bound glycerol (as monoacylglycerides, diacylglycerides and triacylglyerides) in biodiesel was developed, giving results beyond the state of the art of the current standard methodologies. The proposed method is based on anon-line hydrogenation using gas chromatography coupled to flame ionization detection and hydrogen as carrier gas. After sample introduction the hydrogenation takes place on a fused silica pre-column coated with a palladium catalyst. This approach allows an immediate and reliable hydrogenation of vegetable oils and biodiesels from different feedstocks. All glycerides are converted into their saturated analogues, resulting in simplified chromatograms with structurally clearly defined analytes, and increased sensitivity for trace amounts of compounds. The method has been successfully in-house validated and combined uncertainty values have been assigned to the final results, which were less than 8% for free glycerol, the sum of monoacylglycerides, the sum of diacylglycerides and the sum of triacylglycerides.