Crisaborole ointment, 2%, is a non-steroidal, topical anti-inflammatory phosphodiesterase 4 inhibitor for the treatment of mild-to-moderate atopic dermatitis. To date, a specific analytic method of crisaborole in plasma has not been reported. The aim of this study was to develop a rapid, sensitive and robust UHPLC-MS/MS method for the quantitative detection of crisaborole in human plasma by using deuterated crisaborole-d4 as the internal standard (IS). The analyte was well extracted from human plasma with acetonitrile and subsequently eluted with gradient acetonitrile and water in short run time of 3.3 min. Negative electrospray ionization in multiple reaction monitoring mode was employed to acquire the quantification ion pairs of m/z 250.0→118.0 for crisaborole and m/z 254.0→121.9 for IS. The assay met the regulations of the US Food and Drug Administration and the European Medicines Agency for assay validation with a good linearity in the calibration range of 0.20-80 ng/mL. Intra-day and inter-day precision was less than 9.17% and the accuracy was - 2.29%-6.33% across all the quality control samples. The average extraction recovery of analyte and IS was 84.61% and 91.43%, respectively, and consistent over different quality control samples. The fully validated method was successfully used for the drug level measurement in ten healthy Chinese subjects receiving crisaborole ointment. Our novel UPLC-MS/MS assay for the quantification of plasma crisaborole concentrations in human samples may be easily used in clinical practice and help to reveal the pharmacokinetic profiles of crisaborole in Chinese population.Copyright © 2022 Elsevier B.V. All rights reserved.