Abstract: A method of HPLC-MS/MS coupled with ionic liquid aqueous two-phase extraction was proposed for the determination of brodifacoum in urine. An aqueous two-phase system of hydrophilic ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate and NaH2PO4 was used to extract the brodifacoum from the urine samples. The extract was separated on a XDB C18 column by using the mixture of methanol and 0.2% (φ) acetic acidsolution (85+15) as the mobile phase. ESI and MRM were adopted in MS/MS. The linearity range of the mass concentration of brodifacoum was 0.02-2.0 μg·L-1, with the limit of quantification (10S/N) of 0.015 μg·L-1. The recovery rates measured by standard addition method were in the range of 86.8%-92.5%, with intra-day RSD (n=6) in the range of 3.3%-8.1% and the inter-day RSD (n=6) in the range of 6.2%-9.7%. The proposed method was used to measure trace brodifacoum in urine.