Cholangiocarcinoma (CCA) poses a significant health challenge, especially in Asian countries. Late-stage diagnoses limit the availability of curative surgical options, underscoring the crucial role of chemotherapy. However, drug resistance presents a formidable obstacle, resulting in recurrent tumors. Despite the revolutionary impact of proteomics on cancer research, the exploration of drug-resistant mechanisms in CCA remains unexplored. In this study, we established 5-fluorouracil (5-FU)- and gemcitabine-resistant CCA cell lines, KKU-213FR and KKU-213GR, derived from the KKU-213A parental line. These cell lines exhibited robust drug resistance, heightened migration, and invasion capabilities.