Mucus is mainly composed of water (90-95%) and mucins (2-5%), these mucins being high molecular weight macromolecules forming a 3D cross-linked matrix. It makes it a complex non-Newtonian fluid, with viscoplasticity, viscoelasticity, shear-thinning and thixotropy (Lai et al., 2009). These properties were qualitatively and separately tested by different authors. Due to the difficulties to collect samples and the extreme sensibility of the tested materials, the results are widely variable. A complete and intrinsically consistent characterization remains to be done to develop a reliable rheological model for future numerical simulations of mucus displacements in airways, in the case of pathologies such as cystic fibrosis. For this purpose, samples of mucus simulants were tested using a controlled stress rheometer. The material consists of macromolecular colloidal gels at different concentrations in macromolecules to mimic the variability in mucin production depending on the disease state and environmental factors. The rheological properties at rest were investigated using small amplitude oscillatory shear tests as a function of stress amplitude, frequency and temperature. They revealed that mucus simulant behaves as a gel within a defined linear viscoelastic region and as a viscoelastic liquid above the yield stress zone. To characterize the behavior of mucus in response to in vivo shearing induced by cough or by air flows produced by clearance helping devices, steady state flow tests have been performed. The steady state flow curves for various polymer concentrations are well fitted using a Herschel-Bulkley law. To account for the time dependent behavior of mucus, 3 intervals thixotropy tests were also implemented to monitor structure breakdown and buildup processes. The combination of all of these measurements finally design a reliable procedure accounting for the rheological complexities of mucus, that now needs to be applied to real mucus for validations.