Abstract: The regularity of degradation of amoxillin (AMXL) by its reaction with β-lactamase (β-LTM) was studied by HPLC-TOF-MS. SB-C18 column was used as stationary phase in the chromatographic separation, and mixtures of 0.1% (φ) HCOOH solution and acetonitrile in various ratios were used as mobile phase in gradient elution. Mode of scanning under ESI+ was adopted in MS analysis. It was found that: ① for degradation of 10 μg of AMXL, 7 U of β-LTM was necessary in the reaction; ② the optimum temperature for the degradation was found in the range of 15 ℃-30 ℃, giving reaction time of 2.5 h, the main degradation product is amoxilloic acid (abbr. as Ⅰ), and when the temperature is higher than 40 ℃, another product, the decarboxylated amoxilloic acid (abbr. as Ⅱ) is produced; ③ and it was shown that in a milk sample when acidity was adjusted to pH 2-3, Ⅱ was the main product; when at pH 3-4, amount of Ⅰ was increased gradually while amount of Ⅱ was kept nearly unchanged; and AMXL was completely degraded at pH 7 giving mainly Ⅰ.