In the presented report, we have described the silylation reaction between the amide group in acetaminophen and a two most popular trifluoroacetamide-based silylation reagents – BSTFA and MSTFA. Both reagents had a amide groups on structures. An investigation was made through the performance of a set of experiments, GC–MS analysis, and a theoretical study, namely interpretation of mass spectra, presentation of the resonance states of all the involved compounds and SN2 reaction schemes, which was found to be different when BSTFA and MSTFA was applied. The negligible effect of used solvent was also described. The fragmentation of TMS-derivatives (MS spectra) was presented and it has confirmed our previous investigations with silylation of pharmaceuticals, and a general rules of fragmentation patterns. Thanks to this the structure of di-O,O-TMS-acetaminophen was proven.