The pharmacokinetic characteristics of valnemulin in layer chickens were studied after single intravenous, intramuscular, and oral administration at a dose of 15 mg/kg body weight. Plasma samples at certain time points were collected and the drug concentrations in them by ultra high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS). The concentration-time data for each individual were plotted by noncompartmental analysis for the whole three routes. Following intravenous administration, the plasma concentration showed tiny fluctuation. The elimination half-life (T1/2λz), total body clearance (Cl), and area under the plasma concentration-time curve (AUC) were 1.85 ± 0.43 h, 2.2 ± 0.9 L/h, and 7.52 ± 2.46 μg·h/mL, respectively. Following intramuscular administration, the peak concentration (Cmax , 1.40 ± 0.43 μg/mL) was achieved at the time of 0.34 h. A multiple-peak phenomenon existed after oral administration, and the first peak and secondary peak were at 10 min and during 2-4 h, respectively, while the tertiary peak appeared during 5-15 h. The bioavailability (F %) for intramuscular and oral administration was 68.60% and 52.64%, respectively. In present study, the detailed pharmacokinetic profiles showed that this drug is widely distributed and rapidly eliminated, however has a low bioavailability, indicating that valnemulin is likely to be a favorable choice in the clinical practice.