Understanding synaptic function is essential for investigating brain physiology and pathophysiological changes, such as those occurring in neurodegenerative diseases. Synaptosome isolation, which extracts synaptic terminals containing both pre- and post-synaptic components, provides a valuable tool for studying synaptic biology. Previous research suggests that synaptosome preparation from shock-frozen brain tissue, which is the most common source of human brain synaptosomes, generally yields synaptosomes with a lower respiratory rate, indicating potentially reduced biological functionality compared to fresh tissue. However, detailed comparative studies on the impact of freezing on synaptosome quality are lacking. Our research evaluates how fresh and frozen tissue differ in their suitability for synaptosome isolation, focusing on synaptic protein enrichment, non-synaptic contamination, and the preservation of functionality and structural integrity. We compared synaptosomes isolated from mouse brain tissue that was shock-frozen in liquid nitrogen, slowly frozen with a cryoprotective solution, and freshly processed. Western blot analysis and proteomic profiling were used to assess synaptic protein enrichment and non-synaptic contamination, which will be further validated by electron microscopy. Additionally, functionality assays will determine the impact of tissue state on synaptic exocytosis. Overall, this study aims to evaluate the efficacy of isolating synaptosomes from fresh versus frozen mouse brain tissue. Our findings will contribute to optimizing experimental approaches and improving the comparability of human and preclinical studies in synaptic research.