The inappropriate or illegal use of propofol has recently come to the fore as a serious social issue in South Korea. Thus, in spite of its superior potency as a therapeutic drug, propofol was classified as a controlled drug under the purview of Narcotics Control Law in South Korea in February of 2011. Accordingly, the determination of propofol and/or its metabolites in biological specimens is required to prove ingestion. Therefore, to demonstrate chronic ingestion, a quantitative analytical method for propofol-glucuronide in hair was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method was applied to measure propofol-glucuronide in hair samples from 23 propofol abuse suspects and in both pigmented and nonpigmented hair from rats which had ingested propofol. Propofol-glucuronide in hair was extracted in methanol and then filtered and analyzed by LC-MS/MS with electrospray ionization in negative mode. The validation results of selectivity, matrix effect, recovery, linearity, precision and accuracy, and processed sample stability were satisfactory. The limit of detection was 20 pg/10 mg hair and the limit of quantification was 50 pg/10 mg hair. The concentration range of propofol-glucuronide in hair segments from 23 propofol abuse suspects was shown up to 1,410 pg/mg. The animal study demonstrated that the presence of melanin did not affect the deposition of propofol-glucuronide in hair. Thus, we propose propofol-glucuronide in hair as a marker for propofol abuse. This method will be very useful for monitoring the inappropriate use of propofol for both legal and public health aspects.