One of the most significant issues in pharmaceutical industries, prior to commercialization of a pharmaceutical preparation is the "preformulation" stage. However, far too attention has been paid to verification of the software assisted statistical designs in preformulation studies. The main aim of this study was to report a step by step preformulation approach for a semisolid preparation based on a statistical mixture design and to verify the predictions made by the software with an in-vitro efficacy bioassay test. Extreme vertices mixture design (4 factors, 4 levels) was applied for preformulation of a semisolid Povidone Iodine preparation as Water removable ointment using different PolyEthylenGlycoles. Software Assisted (Minitab) analysis was then performed using four practically assessed response values including; Available iodine, viscosity (N index and yield value) and water absorption capacity. Subsequently mixture analysis was performed and finally, an optimized formulation was proposed. The efficacy of this formulation was bio-assayed using microbial tests in-vitro and MIC values were calculated for Escherichia coli, pseudomonaaeruginosa, staphylococcus aureus and Candida albicans. Results indicated the acceptable conformity of the measured responses. Thus, it can be concluded that the proposed design had an adequate power to predict the responses in practice. Stability studies, proved no significant change during the one year study for the optimized formulation. Efficacy was eligible on all tested species and in the case of staphylococcus aureus; the prepared semisolid formulation was even more effective.