Single hair analysis normally requires extensive sample preparation micro scale protocols including time consuming steps like segmentation and extraction. Matrix assisted laser desorption and ionization mass spectrometric imaging (MALDI-MSI) was shown to be an alternative tool in single hair analysis but still questions remain. Therefore an investigation of MALDI-MSI in single hair analysis concerning extraction process, usage of internal standard (IS) and influences on ionization processes were systematically investigated to enable the reliable application to hair analysis. Furthermore, single dose detection, quantitative correlation to single hair and hair strand LC-MS/MS results was performed and performance compared to LC-MS/MS single hair monitoring. MALDI process showed to be independent from natural hair color and not being influenced by the presence of melanin. Ionization was shown to be reproducible along and in between different hair samples. MALDI image intensities in single hair and hair snippets showed good semi-quantitative correlation to zolpidem hair concentrations obtained from validated routine LC-MS/MS methods. MALDI-MSI is superior to LC-MS/MS analysis when a fast, easy and cheap sample preparation is necessary, whereas LC-MS/MS showed higher sensitivity with the ability of single dose detection for zolpidem. MALDI-MSI and LC-MS/MS segmental single hair analysis showed good correlation and are both suitable for consumption monitoring of drugs of abuse with a high time resolution.