The use of cell lines or animal models has significant disadvantages when dealing with a set of heterogeneous diseases such as epithelial ovarian cancer. This has clinical relevance in that biomarkers developed using cell line or animal models are often not transferable to the clinical setting. In this study, we describe the development of a robust protocol for developing primary cultures of ovarian cancer which will overcome some of these difficulties. Women undergoing surgery for ovarian cancer were recruited and samples of ascites and solid tumour deposits were used to develop primary cultures. Cells were characterised using a panel of immunofluorescent antibodies prior to use in a variety of assays including functional assessment of DNA repair pathways. During the four year study period, viable cultures, confirmed to be epithelial in origin were generated from 156 of 172 (91%) cases recruited. Characterisation was carried out using a panel of antibodies including pancytokeratin, CA125, EpCAM, MOC-31, D2-40 and vimentin. Senescence occurred between the 2 nd and 8th passages in all cultures except one in which spontaneous immortalization occurred. Cells could be successfully cultured even after a period of storage at 4°C and cultured cells were capable of being used for a variety of applications including functional assays. Upon functional assessment there was minimal intra-tumour heterogeneity. It is therefore possible to derive viable ovarian cancer cell cultures in the majority of patients undergoing surgery. Cells cultured directly from patient cancers provide an accurate and highly diverse model. © 2014 O9Donnell et al.