Abstract: Synaptosome: Synapses are the most critical portion of neuron connections, necessary for cellular organization of the brain. Synapse integrity is uttermost important for healthy brain functioning. Any perturbation in the synapse structure and/or function initiates neurological disorders. Synapses are the prime targets that are smashed in almost all neurodegenerative diseases. The vital components of synapse are essential for neurotransmission, synaptic plasticity and overall synapse function. Synapse dysfunctions are well studied in Alzheimer’s disease (AD) and other neurodegenerative diseases (Gowda et al., 2021). The best way to study the synapse dysfunctions in neurological diseases is the biochemical analysis of “synaptosome”. Researchers studied the synaptosome to understand the molecular reasons of synapse dysfunction in aging, AD and other neurodegenerative diseases (Kumar et al., 2020; Gowda et al., 2021). Synaptosome maintains the cellular machinery and all vital components necessary for autonomous synapse function. Synaptosome retains mitochondria, synaptic vesicles, lysosomes, endosomes along with the postsynaptic membrane and the postsynaptic density (Lugli et al., 2012; Xu et al., 2013; Li et al., 2015; Kumar et al., 2020). Therefore, synaptosomes hold the molecular machinery necessary for uptake, storage, and release of neurotransmitters, channels, receptors, and local signal transduction. Based on these properties, synaptosomes are called as “Ex vivo” model to study synaptic physiology and pathophysiology. They also pronounced as “halfway house” between neurochemistry and electrophysiology (Lugli et al., 2012; Xu et al., 2013; Li et al., 2015; Kumar et al., 2020). However, there are some technical difficulties while studying the synaptosome. Due to synapse degradation and/or reduced synapse numbers in AD, we need the large amount of brain tissue (≥ 50 mg) to isolate the appropriate quantity of synaptosome for electron microscopy, mRNA and protein analysis. Another technical challenge is mitochondria contamination in synaptosome fraction because of the overlapping size of synaptosome (0.6 to 1 μm) and mitochondria (0.5 to 3 μm). Though mitochondria are a part of synaptosome, but sometimes we observed the separate mitochondrial contamination while preparing the synaptosomes. We can avoid the mitochondria contamination by applying the soft and gentle Dounce homogenization for synaptosome extraction (Kumar et al., 2022).