The prognosis for patients with pancreatic cancer is extremely poor, as they are resistant to first line chemotherapy. The long-term goal of this study was to identify effective combination chemotherapy for pancreatic cancer using pancreatic cancer surgical specimens in the histoculture drug response assay (HDRA) based on three-dimensional culture of tumour fragments, which maintains nature tumour histology in vitro. From 2015 to 2017, the HDRA was performed with tumour specimens from 52 pancreatic cancer patients from Asan Medical Hospital. First, combination drug regimens showed higher drug efficacy and less patient variation than single drugs. Initially, 5-Fluorouracil(5-FU)/Belotecan/Oxaliplatinum and Tegafur/Gimeracil (TS-1)/Oxaliplatinum/Irinotecan were found to be effective. Second, we were able to correlate the efficacy of some drugs with tumour stage. Third, when designing new combination regimens containing 5-FU or gemcitabine, we could identify more effective drug combinations. This is the first study to demonstrate usefulness of the HDRA for pancreatic cancer. Using this technique, we could identify novel candidate combination drug regimens that should be effective in treating pancreatic cancer.