Freeze-drying, or lyophilization, is a widely adopted technique in the pharmaceutical and biomedical fields to enhance the stability and shelf life of sensitive biological products, such as probiotics. These live microorganisms offer numerous health advantages, but they are highly vulnerable to environmental stressors like gastrointestinal conditions, moisture, and temperature. By removing moisture under controlled low-temperature and vacuum conditions, freeze-drying significantly inhibits microbial growth, prevents structural degradation, and preserves cellular integrity of probiotic cells. Ensuring stability of probiotic-based medications and supplements throughout storage and transportation is essential for maintaining their therapeutic efficacy. This chapter also further discusses the significance of the glassy state in minimizing molecular mobility and limiting denaturation of cellular components. Key factors such as integration of cryoprotectants and process optimization (temperature and pressure control) are enhancing bacterial survival, which is required for therapeutic action. Hence, this chapter emphasizes the formulation approaches and novel cryoprotective agents that enhance survivability and functional performance. Overall, freeze-drying remains a key technology for improving probiotic stability, and continuing research focuses on overcoming the related obstacles in order to fully realize its promise in pharmaceutical and biomedical product development.