This chapter describes the methodology and theoretical background for establishing crystallization phase diagrams of biomolecules with a focus on proteins whose crystallization involves specific problems which are not met in the case of small molecules. These problems arise from the effects triggered by the heterogeneity of the protein crystals as a result of water uptake, the drift of pH in the crystallization medium, and the hydrodynamic conditions occurring in stirring devices. Each of them can affect the shape of the phase diagram and hence the location of the crystallization window on the plane of operating variables. Water uptake in protein crystals influences the equilibrium concentration of the constituents of the crystallization medium and, thereby, alters the boundary of the phase diagram. pH drift occurs in the course of the phase transition as a result of protein dissociation in aqueous solutions, which disturbs the crystallization equilibrium. The method of stirring the crystallization medium used to establish the phase diagram has an impact on the formation of the crystalline phase and thus also on the boundaries of the phase diagram. This chapter aims to elucidate the mechanism underlying these effects and provides hints for their quantification and the establishment of crystallization phase diagrams by adequate experimental procedures.