Propofol and dexmedetomidine are both commonly used anaesthetics. Although they employ two different mechanisms to induce anaesthesia, both compounds influence the hippocampus and the HT22 cell line. HT22 cells are broadly used in neurobiological research. In this study, we assessed the effects of propofol and dexmedetomidine on signalling in HT22 cells. Using the SILAC (stable isotope labelling with amino acids in cell culture) labelling technique, IMAC (immobilized metal affinity chromatography) enrichment and high-resolution LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis, we investigated the quantitative proteome and phosphoproteome in HT22 cells treated with propofol or dexmedetomidine. In total, 4,527 proteins and 6,824 phosphosites were quantified in cells treated with these two anaesthetics. With the assistance of intensive bioinformatics, the propofol and dexmedetomidine treatments were shown to induce distinct proteome and phosphoproteome profiles in HT22 cells. Consistent with our bioinformatics analysis, dexmedetomidine had a smaller effect than propofol on cell survival. These findings deepen our understanding of drug-induced anaesthesia.