The invention discloses a method to analyze tobacco glycoside through derivatization, which adopts N-methylene bis (trifluoro acetamide) as the derivatization reagent to derivate the extracted purified tobacco glycoside directly, and then utilizes a GC-MS for qualitative and quantitative analysis. The invention is provided with features like simple design, good reappearance, high accuracy, fast analysis speed etc, and is suitable for simultaneous detection to multiple glycosides components in tobaccos of different types.