A rapid and sensitive method based on ultrafast liquid chromatography–tandem mass spectrometry was developed and validated for simultaneous determination of Sudan I, Sudan II, Sudan III, and Sudan IV levels in rat whole blood. Cleanert C18 mixed-mode polymeric sorbent was used for effective solid-phase extraction cleanup. Separation was carried out on a reversed-phase C18column(100 mm 2.1 mm,1.8 μm) using 0.1%(v/v) formic acid in water/0.1%(v/v) formic acid in acetonitrile as the mobile phase in gradient elution. Quanti fi cation was performed by an electrospray ionization source in the positive multiple reaction monitoring mode using D5-Sudan I as the internal standard. Calibration curves showed good linearity between 0.2 and 20.0 μg/L, with correlation coef fi cients higher than 0.9990. The average recovery rates were between 93.05% and 114.98%. The intra- and inter-day relative standard deviations were within 6.2%. The lower limit of quanti fi cation was 0.2 μg/L. All the analytes were found to be stable in a series of stability studies. The proposed method was successfully applied to a pharmacokinetic study of four Sudan dyes after oral administration to rats. A rapid and sensitive method based on ultrafast liquid chromatography-tandem mass spectrometry was developed and validated for simultaneous determination of Sudan I, Sudan II, Sudan III, and Sudan IV levels in rat whole blood. Cleanert C18 mixed-mode polymeric sorbent was used for effective solid-phase extraction cleanup. Separation was carried out on a reversed phase C18column (100 mm 2.1 mm, 1.8 μm) using 0.1% (v / v) formic acid in water / 0.1% as the mobile phase in gradient elution. Quanti cation was performed by an electrospray ionization source in the positive multiple reaction monitoring mode using D5-Sudan I as internal standard. Calibration curves showed good linearity between 0.2 and 20.0 μg / L, with correlation The average recovery rates were between 93.05% and 114.98%. The intra- and inter-day relative standard deviations were within 6.2%. The lower limit of quanti fi cation was 0.2 μg / L. All the analy tes were found to be stable in a series of stability studies. The proposed method was successfully applied to a pharmacokinetic study of four Sudan dyes after oral administration to rats.