For many cancers, there is a real need for more effective therapies. Although many drugs show promising results in vitro, most fail to translate into an in vivo model system, and only ∼5% show anti-tumor activity in clinical trials. It remains a significant challenge to accurately replicate in vitro the complex in vivo microenvironment in which cancers thrive, but this will be key to increasing the success of translating novel therapies into clinical practice. Three-dimensional (3D) cell culture models may better mimic primary tumors in vivo than traditional two-dimensional (2D) cultures. Therefore, we established and characterized 3D in vitro models of 31 epithelial ovarian cancer (EOC) cell lines, compared their biological and molecular features with 2D cultures and primary tumors, and tested their efficacy as models for evaluating chemoresponse. When cultured in 3D using polyhydroxoethylamethacrylate-coated plastics, EOC lines formed multicellular aggregates that could be classified as 'large dense', 'large loose', and 'small', based on size, light permeability, and proportion of cells incorporated into the complex structures. Features of histological differentiation characteristic of primary tumors that were not present in 2D cultures were restored in 3D. For many cell lines, the transition from a 2D to 3D microenvironment induced changes in the expression of several biomarkers relevant to disease. Generally, EOC cell lines proliferated more slowly and were more chemoresistant in 3D compared with 2D culture. In summary, 3D models of EOCs better reflect the histological, biological, and molecular features of primary tumors than the same cells cultured using traditional 2D techniques; 3D in vitro models also exhibit different sensitivities to chemotherapeutic agents compared with 2D models, which may have a significant impact on the success of drug testing pipelines for EOC. These findings could also impact in vitro modeling approaches and drug development strategies for other solid tumor types.