In this study, MTBSTFA and BSTFA, which are among the preferred derivatization reagents for silylation were both tested on derivatization of six different groups of polar chemicals to get information about usefulness in terms of sensitivity and specificity of both reagents. Tested compound groups were nitrophenols and methoxyphenols, sterols and sugars, dicarboxylic acids and hydroxylated polycyclic aromatic hydrocarbons. It was found that MTBSTFA-derivates produce characteristic fragmentation patterns presenting mainly the fragments [M](+), [M-57](+) and [M-131](+), of which [M-57](+) is generally dominant on the mass spectrogram. BSTFA-derivates mainly show the fragments [M](+), [M-15](+) and [M-89](+) whereof the molecular ion [M](+) is generally dominant. It was also found that steric hindrance and molecular mass play a very important role in the choice of the best suited derivatization reagent: compounds with sterically hindered sites derivatized with MTBSTFA produce very small analytical responses or no signal at all, and compounds with high molecular mass produce no characteristic fragmentation pattern when derivatization is performed with BSTFA. It was also found that MTBSTFA-derivatization facilitates separation of isomer analytes, suggesting its choice in combination to semi-polar columns, whilst BSTFA seems better for sterically hindered compounds. Findings were confirmed with applications of both reagents to biological and environmental matrices (urine and atmospheric aerosols).