From a therapeutic point of view, ALDH activity is important in alcohol metabolism through aldehyde detoxification and to cancer drug resistance through the metabolism of chemotherapeutics such as cyclophosphamides. Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide-range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization and structure-activity-relationship (SAR) studies of small molecule ALDH1A1 inhibitors with cellular activity, this collaborative team worked to develop and miniaturize a high-content cell-based ALDEFLUOR assay. During this period, the project team designed and optimized a new quinoline-based series as potent ALDH1A1 inhibitors, based on previously completed high-throughput screening work. Extensive medicinal chemistry work has been completed with more than 300 analogs generated. This series exhibited excellent cell activity in several cancer cell lines, and selected analogs demonstrated target engagement in a cellular thermal shift assay (CETSA). Additionally, members of this series inhibited the formation of 3D spheroid cultures of OV-90 cancer cells, and potentiated the cytotoxicity of Paclitaxel in SKOV-3-TR, a Taxol-resistant ovarian cancer cell line. Together with appropriate pharmacokinetics (PK), they appear suitable for potential in vivo POC studies.