The microenvironment plays a major role in conferring chemoresistance to cancer cells. In order to better inform clinical response to chemoresistance, preclinical models that recapitulate its hallmark features are needed to enable screening for resistance-specific therapeutic targets. A novel platform for seeding cancer cells in 3D hydrogels is presented utilizing derivatives of chitosan and alginate that, critically, is amenable to high throughput screening: cell seeding in hydrogels, media changes, dosing of anticancer compounds, and cell viability assays were all automated using a standard and commercially available liquid handling robot. Culture in these hydrogels elicits resistance in ovarian, lung, and prostate cancer cells to treatment by doxorubicin and paclitaxel. In correlation, proteomics analysis of SKOV3 cells cultured in 3D revealed enrichment of proteins associated with extreme drug resistance (EDR) including HMOX1 and ALDH2. Subsequently, therapeutic antibodies targeted to tumor-associated antigens upregulated in 3D cultures were shown to have higher efficacy compared to 2D cultures. Collectively, this automated 3D cell culture platform provides a powerful tool with utility in identification of drugs that may overcome chemoresistance. This article is protected by copyright. All rights reserved.