This publication deals with the optimal experimental design for the determination of diffusion and kinetic parameters of hetorogenous biocatalytic reactions employing immobilized whole cells in a batch reactor. A reaction rate and progress curve methods, based on the measurements for a set of particle radii and a single value of initial substrate concentration, were elaborated. The experimental system studied was sucrose hydrolysis catalysed by Saccharomyces cerevisiae cells with invertase activity immobilized in Ca-alginate gel particles, Monte Carlo simulations were employed to analyse the effect of experimental design on the accuracy of the parameter estimates in both methods. The results obtained show that the critical factor of the design was the range of Thiele moduli corresponding, to the different particle sizes which, however, could be adjusted by the choice of a proper value of intraparticle cell concentration. The optimum region of the intraparticle cell concentration obtained from the simulation studies was verified experimentally. Very close values of parameters obtained from three different runs confirmed the quality of the experimental design and, moreover, validated the obtained value of sucrose effective diffusivity.