Short organic acids, especially those with an additional hydroxygroup, are difficult to analyze by GC-MS. BSTFA alone or in combina-tion with other silylating reagents such as TMCS are very commonlyused for the GC-MS analysis carboxylic (COOH) and hydroxylic (OH)groups-containing analytes. Unlike carboxylic groups, aliphaticgroups are quite inert towards silylating reagents, and their conver-sion to volatile TMS derivatives requires quite high derivatizationtemperatures and times. On the other hand, TMS ester-TMS etherderivatives are highly sensitive to humidity, so that considerablehydrolysis may occur. These circumstances are of particular impor-tance when no stable-isotope labeled analogues of selected organicacids are available or they can not be used in routine analysis.In the present paper we report on the development, optimizationand validation of a GC-MS method with electron ionization for thequantitative determination of a set of short organic acids in humanurine in clinical settings. In our study we focused on the optimiza-tion of the derivatization of short organic acids by BSTFA/TMCSand the stability of TMS derivatives. These important issues aresomewhat neglected in the literature so far. Our study indicatesthat silylation of organic acids extracted from urine and stabilityof their TMS derivatives are of particular importance and need tobe implemented in method development. We are now improvingand expanding the GC-MS method to a larger set of organic acids,aiming to support Pediatric clinics in Northern Greece and assist inclinical diagnostics.