With the aim of studying the pharmacokinetics of letermovir, which is a newly developed antiviral agent for human cytomegalovirus, a rapid and simple ultra-performance liquid chromatography coupled with mass spectrometry (UPLC/MS) method was developed and validated for the quantification of letermovir in human plasma. Separation was performed in reverse phase mode using an ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 × 50 mm) at a flow rate of 0.3 mL/min, 10 mM ammonium acetate–0.1% formic acid solution as mobile phase A, and acetonitrile as mobile phase B with a gradient elution. The method was validated over a linear range of 10–1000 ng/mL with a coefficient of determination (R²) >0.99 using weighted linear regression analysis. The intra- and inter-assay accuracy (nominal%) and precision (relative standard deviation%) were within ±15 and ≤15%, respectively. The specificity, recovery, matrix effect, stability, and dilution integrity of this method were also within acceptable limits. This method could be useful in studying the pharmacokinetics and pharmacodynamics, as well as performing the therapeutic drug monitoring of letermovir.