Freeze-drying of pharmaceuticals, especially large molecules, has become increasingly important in recent years. Even though freeze-drying is generally considered a gentle drying process, freezing and drying conditions can affect the stability and activity of many APIs, especially proteins. The present work investigates the impact of the freezing protocol and, hence, of the structure of freeze-dried products on the activity of a model protein, i.e., lactate dehydrogenase. Using a systematic approach, the main cause of stress during the freezing and drying steps were analysed for three different freezing methods, precooled-shelf, shelf-ramped, and vacuum-induced surface freezing. As each freezing protocol led to the formation of ice crystals of different dimensions, it was found to influence the loss of activity during both freezing and drying.