We present a protocol for the fabrication of reusable inserts for standard 24-well plates, comprising a 3D-printed mounter and a hydrogel scaffold for 3D cell culture studies. We cover the design and production of the mounter, hydrogel scaffold synthesis, lyophilization for storage, sterilization, and hydrophilization before cell culture. Downstream applications include cytotoxicity assessment, imaging, and qPCR. Streamlining hydrogel preparation and mounting minimizes batch-to-batch variation, reduces costs, and supports synthetic biology studies.