Abstract: Introduction: Biomacromolecules have received increased focus in the pharmaceutical field during recent years. In order to overcome their low stability, these molecules are commonly freeze-dried along with stabilizers and bulking agents. The solid form of the common bulking agent mannitol has been reported to affect the stability of freeze-dried samples. As processing conditions affect the solid form of mannitol, the effect of changing freeze drying parameters was investigated, applying in-situ freeze-drying. Materials and Mthods: In-situ freeze-drying was carried out in the temperature stage of a variable temperature X-ray powder diffractometer, connected to a vacuum pump. 100 μl of sample solution containing mannitol along with protein or the stabilizer sucrose was placed in the sample holder. The samples were frozen at different rates and dried applying vacuum to the temperature chamber, continuously recording diffractograms during the process. Results and Discussion: Freezing and annealing had the most pronounced effect on mannitol solid state during the freeze-drying process. Intermediate freezing rates caused formation of hemi-hydrate, while fast and slow freezing caused no mannitol crystallization. Formation of d-mannitol during subsequent annealing was found not to be a conversion of hemi-hydrate but rather a crystallization process from amorphous mannitol. Generally no change in mannitol solid state was seen during primary drying. Secondary drying lead to conversion of mannitol hemi-hydrate to d-mannitol. Preforming secondary drying at 30°C had little effect on the hemi-hydrate, while drying at 40°C for three hours lead to almost complete conversion of hemi-hydrate. Conclusion: In-situ freeze-drying was successfully used to provide insight into the solid state behavior of mannitol during the different stages of freeze-drying.