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Oral administration of colon-targeting nanoformulations holds many advantages over the systemic delivery of free drugs, or traditional nontargeting formulations in the treatment of ulcerative colitis (UC). Currently, the most conventional method for constructing colon-targeting drug delivery systems (DDS) is by integrating the biocompatible materials poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) into a copolymer. This PLGA/PLA-polyethylene glycol-folic acid (PEG-FA) copolymeric nanoformulation effectively delivers the drugs for uptake by various human colon cancer cells (e.g., HT-29 and HCT-116) and mouse colon cancer cells (CT-26). There is, however, a distinct lack of comprehensive protocols for the construction of such copolymer. This protocol details an easy-to-follow single-step method for the construction of a colon-targeting PLGA/PLA-PEG-FA nanoformulation, which encapsulates a fluorescent dye and demonstrates the visualization of its cell uptake in vitro.