The aim of this study was the development of a method for the simultaneous determination and differentiation of fatty acid esters of 3-monochloropropane-1,2-diol (3-MCPD esters) and glycidol (glycidyl esters) in different foodstuffs. The esters were isolated from fat-rich food samples using a single extraction step and separated from interfering substances. For differentiation of 3-MCPD esters and glycidyl esters the glycidol moiety was converted into 3-methoxypropane-1,2-diol (3-MPD) by acidic alcoholysis. Subsequent determination was achieved by isotope dilution GC-MS after transesterification using an isotope-labeled 3-MCPD ester as internal standard. During optimization of the procedure, critical parameters affecting simultaneous determination and differentiation of these analytes were investigated. Rapid ester cleavage and derivatization at ambient temperature proved to be essential for the simultaneous determination of these analytes. The method was validated for various fat-rich foodstuffs such as bakery products, sweets, gravy, and soup powders as well as edible fats and oils. LODs of 8 and 15 μg/kg (fat-rich foodstuffs) as well as 50 and 65 μg/kg (edible oils and fats) were obtained for 3-MCPD esters and glycidyl esters, respectively. Recoveries for 3-MCPD esters and glycidyl esters ranged within 98 ± 4 and 88 ± 2% in all tested foodstuffs (0.05-2.5 mg/kg) and within 99 ± 16 and 93 ± 13% for edible oils and fats (0.15-3 mg/kg) over a wide concentration range. These results proved an accurate and differentiated determination of 3-MCPD esters and glycidyl esters with successful application to the fast screening of samples, avoiding tedious and laborious sample preparation.