Objective To set up a determination method of warfarin,coumatertralyl,bromadiolone and brodifacoum.Methods HPLC-ESI-MS/MS was used to detect warfarin,coumatertralyl,bromadiolone and brodifacoum using ZORBAX sb-aq(1 50mm×2.1 mm×3.5 μm)as chromatographic column at constant temperature 30℃ while methanol of 0.1%formic acid and water as mobile phase in the grads program.The flow rate was 0.4 mL/min;Detection was performed on an electrospray ionization(ESI)in the negative model and the MRM condition.Results The limit of qualitation was found to be less than 40 pg for four raticides at one time and 10pg for every raticide in the MRM condition;The four raticides linear range were suitable and r were more than 0.999.Matrix affection Was also determined.Conclusion This method is simple,sensitive and accurate for the determination of warfarin,coumatertralyl,bromadiolonc and brodifacoum in the biological tissue.