Abstract A high throughput screening method was developed for the simultaneous analysis of twenty representative tranquilizers in dairy products using the combination of ultra-performance liquid chromatography-high resolution time-of-flight mass spectrometry and screening database built with Target Analysis software. The protein and fat in the sample was removed using a two-step precipitation of acetonitrile and acidic acetonitrile, and the supernatant was combined and concentrated with Speedvac concentrator. The chromatographic separation was performed on an ACQUITY BEH column with gradient elution using 0.1% formic acid and acetonitrile as mobile phase at a flow rate of 0.3 mL min −1 . The monitoring of tranquilizers was achieved by time-of-flight mass spectrometry under positive ionization mode in 9 min. The quantification was performed with matrix-matching method. The linear ranges were 1–500 μg L −1 or 5–1000 μg L −1 . The LODs and LOQs were 0.3–1.5 μg L −1 and 1–5 μg L −1 , respectively. At 5 and 50μg L −1 spiked levels, the average recoveries were 76.1%–108.2% with relative standard deviations of 2.5%–9.0%. The screening result for spiked sample shows all the spiked tranquilizers could be correctly identified with low deviations of retention time ( 89.5%). The developed method was further applied for the analysis of 50 real dairy products, and no positive sample was detected.