Recently, ionic liquids (ILs) are finding ever broader scope within pharmaceutical and bioanalytical applications. In the current work, ACE binding measurements of tryptophan (Try)-HSA, chlorambucil (CHL)-HSA, and dacarbazine (DTIC)-HSA complexes were estimated in the absence or presence of several short chain imidazolium ILs within the range of concentrations of 10.0-1000.0 mu mol/L that are far below the critical micelle concentrations of ILs. Results indicated that the value of binding constant of Trp-HSA was dramatically deviated in the presence of 1000.0 mu mol/L 1-decyl-3-methylimidazolium bromide (DMIMBr) IL. However, interestingly, there is no any deviation for the Trp-HSA binding constant with 100.0 mu mol/L 1-butyl-3-methylimidazolium bromide (BMIMBr) IL as an adjuvant additive in 67.0 mmol/L phosphate buffer at pH 7.4. This finding was further used to estimate the binding constants of important but weakly binding substances of CHL and DTIC antitumors with HSA; their binding constants were also estimated by HPAC giving data in good agreement with that revealed by ACE. These achievements were attributed to the significant improvement of HSA stability by combination with BMIMBr IL through hydrogen bond, electrostatic, and pi-pi forces. In addition, the use of 100.0 mu mol/L BMIMBr extended the stability of native HSA solution stored under the ambient lab conditions up to 25 days with significant improvements in the precision of ACE binding data.