This study was to investigate the effects of different freeze–drying factors on the rate of sublimation. The experiments were carried out in a custom-built freeze–drying microbalance to accurately monitor the sample temperature and control the chamber pressure. Twenty-four experiments were conducted based on a full factorial design by changing four factors: freezing rate (fast freezing or slow freezing), chamber temperature (35, 0, or −35 °C), chamber pressure (30 or 1000 mTorr), and the presence or absence of an annealing process. Lactate dehydrogenase (LDH), a tetrameric protein, was selected as a model protein for this study. The statistical analysis of the experimental results revealed that chamber temperature, analogous to the shelf temperature, in this experiment system, had the greatest impact on the sublimation rate. High chamber temperature resulted in high sublimation rate, regardless of the chamber pressure and thermal history of the sample. Chamber pressure was an important factor affecting the sublimation rate. In addition, both chamber temperature and chamber pressure had significant impact on sample temperature during freeze–drying. Annealing the samples was the most critical step to preserve good freeze–dried cake structure.