Contemporary challenges in probiotic research encompass the development of advanced food products, dietary supplements, and therapeutic formulations that adhere to regulatory standards for safety, practicality, and efficacy. While numerous strains of lactic acid bacteria have been shown to possess probiotic properties and are extensively employed in supplements, ongoing investigations aim to identify novel strains with potentially enhanced health-promoting effects. In vivo models are widely regarded as the gold standard for evaluating probiotic safety. These studies play a crucial role in elucidating the fate of probiotics and their bioactive components following oral administration. Accordingly, this chapter focuses on the preclinical assessment of the safety and toxicity of probiotic organisms, presenting a comprehensive analysis of various in vivo acute and chronic toxicity assessment studies conducted using appropriate animal models.