<p>This study investigates the phase behavior of Poloxamer 188 (P188) in aqueous solutions under conditions typical of pharmaceutical lyophilization processes. We utilized heat flux sensors (HFSs) in a freeze-dryer and replicated lyophilization processes using a miniaturized setup in an XRPD climate chamber. HFS data from vials in the freeze-dryer indicated not only the primary crystallization of ice but also detected the secondary crystallization of the P188-water system. This was possible when primary ice nucleation was well-controlled and consistent across all vials, ensuring that primary crystallization signals didn’t overshadow those from secondary crystallization. Our findings reveal that P188 crystallization in aqueous systems is generally slow. Measurements in the XRPD climate chamber, involving a P188-based placebo solution, demonstrated that only a small portion of P188 crystallizes during the annealing step. The majority of P188 crystallizes during the primary drying step, concurrently with ice sublimation. Additional crystallization of P188 is observed even at the final secondary drying stage. These insights provide a valuable foundation for optimizing industrial lyophilization processes for Poloxamer-based lyophilizates.</p> Graphical Abstract <p>This study explores Poloxamer 188 crystallization during typical freeze-drying conditions for biologics. Heat flux sensors and an XRPD climate chamber reveal that P188 crystallizes quite slowly. Insights inform optimization of lyophilization processes of P188-containing formulations.</p> <p></p>