Gastrointestinal stromal tumors are rare mesenchymal tumors for which complete surgical resection is the only curative treatment, but intraoperative discrimination between tumor and healthy tissue remains chal-lenging. This study aimed to identify molecular biomarkers suitable for fluorescence image-guided surgery in gastrointestinal stromal tumors beyond KIT and DOG1. Biomarker candidates were identified through integrated database mining (Euretos, Open Targets, and clinical trial registries). Candidate selection was refined based on overlap across datasets and membrane localization, resulting in selection of CD243 (ABCB1), PDE3A, PIK3CG, DDR2, and TEM1. Immunohistochemistry was performed on 23 patient-derived tumor samples with matched adjacent tissue, and tumor-to-normal ratios and expression patterns were analyzed. CD243 and PDE3A showed the most favorable tumor-to-normal ratios across multiple tissue types, while PIK3CG demonstrated intermedi-ate expression. TEM1 and DDR2 showed more variable expression and lower overall scoring. A composite scor ing system integrating expression patterns and tumor specificity identified CD243 and PDE3A as leading candi-dates. These results suggest that CD243 and PDE3A are promising targets for further development of fluores-cence-guided surgery in gastrointestinal stromal tumors, warranting validation in preclinical imaging models and clinical translation studies. Simple Summary Gastrointestinal stromal tumors are rare cancers of the digestive tract that are usually treated with surgery. However, it is often difficult for surgeons to clearly distinguish tumor tissue from healthy tissue during an operation, due to minimal tactile feedback and intraluminal tumor depositions. Especially in patients with metastasized disease which can lead to incomplete removal or unnecessary removal of normal tissue. Fluo-rescence-guided surgery, in which tumor targeting molecules are linked to a fluorescent signal, may improve tumor visibility during surgery. In this study, we searched multiple biological databases and combined the re-sults with protein data from tumor tissues to identify potential molecular targets in gastrointestinal stromal tumors. We then validated several candidate targets in patient tumor samples using laboratory staining techniques. We identified several proteins that are highly expressed in tumor tissue compared to normal tissue, with some showing favorable characteristics for imaging applications. These findings may help develop new imaging tools to improve the precision of surgery for patients with gastrointestinal stromal tumors.