We present a method for the quantitation of motilin from rat plasma by protein precipitation and liquid chromatography/mass spectrometry (LC/MS). Using external calibration, the method was linear over the concentration range 10–1000 ng/mL with an initial sample volume of 150 μL. The LC system included a C18 column with a 300 Å pore size. A linear gradient was used with a mobile phase consisting of water and acetonitrile, each with 0.2% acetic acid and 0.02% trifluoroacetic acid. Motilin was detected with the mass spectrometer in positive ion mode monitoring the 4+ charge state at m/z 675.5. The approximated limit of detection was less than 1 ng/mL and the lower limit of quantitation (LLOQ) was 10 ng/mL. The method showed a high degree of precision and accuracy both within and between runs at five validation points, including the LLOQ. Copyright © 2004 John Wiley & Sons, Ltd.