Synapses are essential units for the flow of information in the brain. Over the last 70 years, synapses have been widely studied in multiple animal models including worms, fruit flies, and rodents. In comparison, the study of human synapses has evolved significantly slower, mainly because of technical limitations. However, three novel methods allowing the analysis of molecular, morphological, and functional properties of human synapses may expand our knowledge of the human brain. Here, we briefly describe these methods, and evaluate how the information provided by each unique approach may contribute to the functional and anatomical analysis of the synaptic component of human brain circuitries. In particular, using tissue from cryopreserved human brains, synaptic plasticity can be studied in isolated synaptosomes by fluorescence analysis of single-synapse long-term potentiation (FASS-LTP), and subpopulations of synapses can be thoroughly assessed in the ribbons of brain tissue by array tomography (AT). Currently, it is also possible to quantify synaptic density in the living human brain by positron emission tomography (PET), using a novel synaptic radio-ligand. Overall, data provided by FASS-LTP, AT, and PET may significantly contribute to the global understanding of synaptic structure and function in both healthy and diseased human brains, thus directly impacting translational research.