Patient-derived neurons generated from human induced pluripotent stem cells (iPSCs) form an essential cornerstone for neurodegenerative disease research. Unfortunately, cell cultures in conventional Petri dishes cannot resemble complex in vivo conditions, and also brain-on-a-chip (BoC) approaches in two-dimensional microfluidic devices do not reflect the three-dimensional nature of the human brain. However, state-of-the-art microfabrication techniques employing two-photon lithography (TPL) allow for high-precision prints of 3D scaffolds for cell culturing. In this contribution, we demonstrate that human iPSC-derived neurons can be cultivated and guided in 3D-printed scaffolds to form tailor-made neuronal networks. Not only the electrophysiologically mature state of the neurons was proven by patch-clamp measurements of action potentials but also synaptic activity—a prerequisite for functional network activity—is captured by spontaneous excitatory postsynaptic currents. In our routine, pre-mature neurons are transferred to the platform and the networks are formed within only a few days. In combination with the versatile nature of TPL to create any shape of 3D scaffold to increase the complexity of network topology, we believe that our studies employing patient-derived human neurons deliver novel approaches valuable in human disease research.