The binding constants (Kb) of four novel anti-HIV-1 active compounds with bovine serum albumin (BSA) were determined by capillary zone electrophoresis (CZE) with UV detection under condition of phosphate buffer (pH 8.0, 50 mmol/L) at 15 kV running voltage. These molecules were synthesized by computer-simulated design with the property of inhibiting the binding of HIV-1 Tat protein to trans-activation response region (TAR) RNA required for HIV-1 transcription and blocking the HIV replication cycle. The results showed that with the addition of different concentrations of BSA into the buffer solution, the binding constants of four active compounds (IG3, iso-C3, C3, MC3) with BSA could be measured by the change of migration time. The experimental values of Kb were 1.07 x 10(4), 1.34 x 10(4), 8.51 x 10(3) and 9.45 x 10(3) L/mol, respectively. It is an easy and simple method to estimate the interaction of small molecules with biomacromolecules with 1:1 molar binding ratio.