Alprazolam and CGS21680 are both drugs known for their sleep and sedation effects. The study aimed to utilize mass spectrometry imaging to assess the regulatory changes in brain lipids of mice by the sleep-enhancing effects of the co-administration of alprazolam and CGS21680. A seven-day continuous administration mouse model was established using alprazolam and CGS21680. Glycerophosphoinositols were elevated in nine regions, glycerophosphoglycerols rising in six brain regions, and glycerophosphoserines increasing in six regions. Other lipid classes exhibited reductions, with fatty acids decreasing in 10 regions, carnitines in seven regions, ceramides in nine regions, and diacylglycerols in 10 regions. The research provides valuable insights into sleep-related regulation pathways by delineating the regulation of various lipid biomarkers in multiple brain regions.