Cytoskeletal networks are 3D arrangements of filaments whose complex spatial structure contributes significantly to their intracellular functions, e.g. biomechanics and cargo motility. Microtubule networks in cells are a particular challenge for in vitro modeling because they are sparse and possess overall structure and so cannot be approximated experimentally as a random hydrogel. We have used holographic optical trapping to precisely position and hold multiple microtubule filaments in an in vitro assay, where chemical and environmental variables can be carefully controlled. Below we describe the relevant practical details of the approach and demonstrate how our approach can scale to accommodate modeling of molecular motor transport and biomechanics experiments.