In this work, the interaction of a series of acetylcholinesterase inhibitors (AChEIs; donepezil, galanthamine, huperzine and neostigmine) with human serum albumin (HSA) immobilized on porous silica particles was studied using a biochromatographic approach. For all the tested AChEI molecules, linear retention plots were observed at all temperatures. An analysis of the thermodynamics (i.e. enthalpy (ΔH°), entropy ((S°*)) of the interaction of the AChEI molecules with the immobilized human serum albumin was also carried out. The (H° and (S°* values for donepezil, galanthamine and neostigmine, were negative due to van der Waals interactions and hydrogen bonding which govern this association with albumin. Whereas the positive values of (H° and (S°* of huperzine binding on HSA indicated a predominance of hydrophobic interactions. The association of AChEIs with HSA was increased linearly with pH. A comparative thermodynamic study with benzodiazepine molecules was also done to determine the potential binding site of these drugs on HSA.