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 C18 column (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. Quantification 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 coefficients 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 quantification was 0.2 μg/L. All the analytes were found to be stable in aseries of stability studies. The proposed method was successfully applied to a pharmacokinetic study of four Sudan dyes after oral administration to rats.