The determination of the degree of substitution (DS) of fatty acid cellulose esters with alkyl chain lengths from C8 to C18 was performed by direct transesterification with trimethylsulphonium hydroxide (TMSH) using tert-butyl methyl ether (MTBE) as a solvent. Transesterification was demonstrated to be quantitative at 75 °C in 60 min. The quantification of the formed fatty acid methyl esters was performed by gas chromatography (GC). After the optimization of the method, long chain cellulose esters (LCCE) could be analyzed in a wide range of DS. The obtained values were compared to those given by other existing protocols. LCCE with DS-values in a range of 5 × 10−5 to 3 were analyzed with high accuracy. Reproducibility is weakened for high DS values if the sample has a compact aspect limiting the accessibility of TMSH to the ester functions. This method can also be suitable for the analysis of mixed cellulose esters.