In order to investigate the incorporation of drugs into hair, matrix-assisted laser desorption/ionization-time-of-flight tandem mass spectrometry (MS/MS) imaging was performed on the longitudinal sections of single scalp hair shafts sampled from volunteers after a single oral administration of methoxyphenamine (MOP), a noncontrolled analogue of methamphetamine. Hair specimens were collected by plucking out with the roots intact, and these specimens were prepped by an optimized procedure based on freeze-sectioning to detect the drug inside the hair shaft and hair root. Time-course changes in the imaging results, with confirmatory quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for each 1-mm segment of single hair strands, revealed a substantial concentration of the drug first onto the hair bulbs after ingestion, while only a small portion appeared to be incorporated into the hair matrix, forming a 2-3 mm distinctive drug band with tailing. Comparable amount of the drug also appeared to be incorporated into the keratinized hair shaft in the upper dermis zone, forming another distinct drug band of about 2 mm, which both moved toward the distal side, following the strand's growth rate. These findings provide forensically crucial information: there are two major drug incorporation sites, at least for MOP, which cause overlap of the recordings and deteriorates its chronological resolution down to about 11 days or perhaps longer.