The abnormally thick glycocalyx of cancer cells can provide a physical barrier to immune cell recognition and effective immunotherapy. Here, we demonstrate an optical method based on Scanning Angle Interference Microscopy (SAIM) for the screening of therapeutic agents that can disrupt the glycocalyx layer as a strategy to improve anti-cancer immune responses. We developed a new membrane labeling strategy utilizing leucine zipper pairs to fluorescently mark the glycocalyx layer boundary for precise and robust measurement of glycocalyx thickness with SAIM. Using this platform, we evaluated the effects of glycosylation inhibitors and targeted enzymatic degraders of the glycocalyx, with particular focus on strategies for cholangiocarcinoma (CCA), a highly lethal malignancy with limited therapeutic options. We found that CCA had the highest mean expression of the cancer-associated mucin, MUC1, across all cancers represented in the cancer cell line encyclopedia. Pharmacological inhibitors of mucin-type O-glycosylation and mucin-specific proteases, such as StcE, could dramatically reduce the glycocalyx layer in the YSCCC model of intrahepatic CCA. Motivated by these findings, we engineered Natural Killer (NK) cells tethered with StcE to enhance NK cell-mediated cytotoxicity against CCA. In a CCA xenograft model, these engineered NK cells demonstrated superior anti-tumor efficacy compared to wild-type NK cells, with no observable adverse effects. Our findings not only provide a reliable imaging-based screening platform for evaluating glycocalyx-targeting pharmacological interventions but also offer mechanistic insights into how CCA may avoid immune elimination through fortification of the glycocalyx layer with mucins. Additionally, this work presents a novel therapeutic strategy for mucin-overexpressing cancers, potentially improving immunotherapy efficacy across various cancer types.