Propolis is composed of several bioactive compounds, which give it many beneficial biological effects. The use of cyclodextrins (CDs) is necessary aiming to enhance the stability, absorption, and solubility of these substances in physiological media. The purpose of the present work is to study solubility diagrams of propolis extract of Melbou region (PEM) in the presence of CDs: α‐CD, β‐CD and PM‐βCD, then to formulate an optimized preparation of the inclusion complex using a D‐optimal experimental design with three factors (ratio, ethanol solvent, and temperature). The optimal formulation was characterized by infrared spectroscopy (FTIR), x‐ray diffraction (XRD), and scanning electron microscopy (SEM). The cytotoxic and protective effects of optimal formulation were evaluated in vitro against Human red blood cells (RBCs) and beef sperms. The results showed that PM‐βCD was more efficient, this is confirmed with an optimal formulation that obtained using 40 ml of solvent, a temperature of 40°C with 1:1 stoichiometry. The cytotoxicity tests revealed that PEM and itsinclusion complex are non‐toxic to RBCs and could maintain sperm conservation.It is concluded that PM‐βCD is more suitable to improve the solubility and stability of PEM constituents, and their non‐toxic effect has been approved.This article is protected by copyright. All rights reserved