Abstract: HPLC-MS/MS was applied to the determination of methyl isothiazolin ketone and its chloro-derivatives in perfume and essential additives of tobacco. The sample was extracted with methanol, and the extract was separated on Agilent ZORBAX 300SB-C8 column, using mixtures of methanol and water (mixed in different ratios) as mobile phase in gradient elution. ESI+ and multi-reaction monitoring mode were adopted in MS/MS determination. Linearity ranges of methyl isothiazolin ketone and its chloro-derivatives were found same between 0.05 mg·L-1 and 5.0 mg·L-1, with detection limits of 3.2, 3.6 μg·L-1 respectively. Values of recovery found by standard addition method were in the range of 77.1%-95.0%, and values of RSD′s (n=6) no more than 5.0%.