Individuals in any profession can succumb to chemical abuse. Among the healthcare profession, nurses represent a specific group because of their ease of access to drugs, particularly narcotics. Opioids, potentially highly addictive agents, are usually their drug of choice. Nalbuphine, a synthetic opioid analgesic, is prescribed for moderate-to-severe acute pain, for chronic pain syndromes, and in obstetrics to decrease the adverse respiratory effect of opioid epidural administration. The case of a nurse who was suspected of drug misuse after the disappearance of two nalbuphine ampules in an obstetrics service is described. Because of discrepancies in the results of her blood and urine samples, a sample of head hair was subsequently collected from the nurse. A hair analysis of nalbuphine by liquid chromatography-mass spectrometry has not been previously described. Following decontamination and grinding, hair was mixed with a Söerensen buffer, then subjected to ultrasonic treatment (1 h), and extracted with ethyl acetate. A quantitative analysis was performed with two channels (30 and 45 V), and it is based on a m/z 358 for nalbuphine and a m/z 330 for methylclonazepam as an internal standard. The method was linear from 0.020 to 12 ng/mg of hair (R(2) = 0.972), and the limit of detection and limit of quantitation are 0.020 ng/mg. Accuracy (CV), assessed at 0.4 and 1.6 ng/mg of hair, was 6.18% and 5.77%, respectively, for intraday assays and 4.5% and 10.9% for interday assays. Recovery efficiency at 1.6 ng/mg and 8 ng/mg of hair was 100% and 97.4%, respectively. The hair specimen from the nurse (6 cm) was cut into three equal lengths. Nalbuphine, venlafaxine, and nordiazepam were detected. The concentration of nalbuphine was similar in the three hair locks: 5.07, 7.06, and 5.70 ng/mg of hair. A hair analysis revealed the repeated intake of nalbuphine by the nurse. This person was treated for depression for several months with Effexor (venlafaxine) and Nordaz (nordiazepam) prior to the investigation. Hair appears to be a unique matrix to provide evidence for chronic drug exposure by establishing a historic record that is not possible by blood or urine analysis.