Long before the synapse was first visualized Cajal theorized that dendrites and axons communicate by contacts he called “protoplasmic kisses.” Now, the synapse is a central topic of neuroscience research with thousands of publications each year focused on understanding how synapses function. Isolating synapses by making synaptosomes has become an important tool for studying synapses. The synaptosome is a “halfway house” between neurochemistry and neurophysiology that strips away everything except the synapse, and its molecular machinery to function. This book introduces the reader to a collection of protocols for making synaptosomes. The chapter covers the history of developing synaptosome protocols and 15 detailed protocols for making and using synaptosomes in modern neuroscience research. Here the protocols cover the spectrum from crude synaptic fractions to highly purified functional synaptosomes. Importantly, each chapter describes how the authors have used synaptosomes to address current research questions and gives insights about applying synaptosome protocols to answer new questions about the synapse.