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The Hippo signaling cascade is a major pathway that integrates a broad spectrum of mechanosensory signals at the plasma membrane and regulates response via control of cell proliferation, self-renewal, differentiation, and apoptosis. Dysregulation of this pathway has been observed across a range of cancer types and results in an altered activity of its primary downstream effectors, the oncogenic transcription factors YAP/TAZ. For example, both germline and somatic loss-of-function mutations in the tumor suppressor gene NF2, a component of Hippo, induce hyperactivation of YAP/TAZ, transcriptional changes and ultimately result in tumor growth. The Hippo signaling pathway is an attractive target for drug discovery efforts, however, it is highly complex and still incompletely understood. Hence it is indispensable to get a deeper insight into the Hippo - YAP/TAZ signaling axis. To this end, we performed a genome-wide CRISPR knockout screen in the triple-negative breast cancer (TNBC) cell line MDA-MB231 (NF2LOF) expressing a YAP/TAZ reporter construct. We identified both negative and positive regulators of YAP/TAZ in breast cancer cells. In a second step, screening hits were further characterized in a focused single-cell CRISPR screen (Perturb-Seq), aiming at better understanding of the effects on YAP/TAZ activity regulation and downstream effects on gene expression. Here we present the technical details of our screening approaches and the results of perturbing known and novel regulators of YAP/TAZ on single cell level. We discuss the use of Perturb-Seq in the initial validation of hits from genome-wide screens and provide data that may serve as a basis for future drug discovery efforts, seeking for novel and effective treatments for triple-negative breast cancers and other malignancies with Hippo pathway alterations. Citation Format: Mareike Berlak, Zuzanna Makowska, Filippos Klironomos, Julia Kuehnlenz, Atanas Kamburov, Andreas Steffen, Martin Lange, Barbara Nicke, Ralf Lesche, Peter Staller, Charlotte Kopitz, Jan Naujoks. Identification of novel YAP/TAZ pathway regulators in the triple-negative breast cancer cell line MDA-MB231 using single-cell CRISPR screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6089.