Abstract A liquid chromatography–electrospray mass spectrometry (LC–ES-MS) was developed for the quantitation of ceftiofur in milk at the 50 ppb tolerance level set by the US Food and Drug Administration (FDA) for the drug. The method used ultrafiltration as a simple and rapid means to prepare the sample for analysis. A 100 μl volume of ultrafiltrate containing ceftiofur was concentrated on-column for LC–MS analysis. The LC separation was accomplished using an acetonitrile gradient with the ion-pair reagent heptafluorobutyric acid (HFBA). Propionic acid was added after the LC column to minimize electrospray signal suppression, enhancing the response for ceftiofur by a factor of 10. The transmission ions from the electrospray interface to the MS was enhanced by a factor of 7 by using a Rf ion guide. The development method could detect ceftiofur to 10 ppb and quantitate the antibiotic from 25–200 ppb (linear correlation coefficient of 0.993). The analysis indicated that bovine milk collected 32 h after dosing with ceftiofur was above the FDA tolerance of 50 ppb, while milk collected 48 h after dosing was found to contain 24–31 ppb of ceftiofur.