A simple, sensitive and reproducible high-performance liquid chromatography method was developed and validated for the determination of 14-O-[(2-amino-1,3,4-thiadiazol-5-yl) thioacetyl] mutilin (ATTM), a new synthesized pleuromutilin derivative with potent antibacterial activity, in broiler chicken plasma after a single intravenous (i.v.), intramuscular (i.m.) or oral (p.o.) administration. Satisfactory separation was achieved on a ZORBAX Ecliplus C18 column (250 × 4.6, 5 μm) with UV detection at 279 nm, using a mobile phase comprising acetonitrile and ultrapure water (50:50, v/v). The elution was isocratic at ambient temperature with a flow rate of 1.0 mL/min. The method exhibited good linearity (R2 > 0.999) over the assayed concentration range (0.12-120.00 μg/mL) and demonstrated good intra- and inter-day precision and accuracy. The method was validated and successfully applied to the pharmacokinetic study of ATTM in chicken plasma after i.v. and p.o. administration.