Ractopamine belongs to a group of beta-agonist compounds. It is a forbidden food additive in most countries because of the many reported collective intoxication outbreaks in humans. Because the drug can be rapidly metabolized and eliminated from animal body, the objective of this study was to develop an efficient and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) analytical method for the detection of ractopamine with cutoff below 1 ng/mL to extend the detection window after withdrawal of the drug and at the same time to meet the requirements of zero-tolerance policy set by most countries. After solid-phase cleanup of the non-hydrolyzed and hydrolyzed urine samples, the residues were dissolved in methanol and analyzed directly by LC-MS-MS. Two transitions were monitored and one ratio determined. Samples reported as positive were required to have the ratio of the transitions within 20% of that determined from known calibration standards. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion has the potential of limiting the sensitivity of the assay. The limits of detection and quantitation of the assay were 0.1 and 0.25 ng/mL, respectively. The intraday precisions at 0.25 and 35 ng/mL were 4.88% and 0.95%, respectively. Interday precisions were 5.64% and 0.9% at the same concentrations. The percentage recoveries at 0.25 and 0.5 ng/mL were 117.8% and 108.4%, respectively.