Precise and flexible control of gene expression is essential for dissecting gene function in complex biological systems. Although recent developments in genetic engineering and CRISPR/Cas9 technology have expanded tools for gene activation, suppression and editing, their application in physiologically relevant models remains challenging, time consuming, and expensive. Here, we present a modular, doxycycline-inducible vector system that integrates gene overexpression, shRNA-mediated knockdown, and CRISPR/Cas9-mediated regulation within a single, lentivirus-compatible system. The modular design allows rapid exchange of selection markers, epitope tags and reporters via Gateway cloning, providing broad adaptability across experimental settings. In addition to standard fluorescent and luminescent reporters, the system includes advanced sensors, such as FUCCI cell cycle reporters, to enable monitoring of cellular processes. By combining fluorescence barcoding with combinatorial genetic perturbations, the platform supports multiplexed analysis of gene function and genetic interactions through phenotypic characterization by multiplex fluorescence imaging or flow cytometry. We demonstrate its utility in vivo with breast cancer intraductal xenografts to reveal that ER+ breast cancer cells (MCF7) rely on androgen (AR), estrogen (ER) and progesterone receptors (PR) for in vivo growth. This versatile gene perturbation system provides tight temporal control, streamlined implementation, and high-content phenotyping capacity facilitating efficient in vitro and in vivo studies while reducing the use of animals in in vivo validation experiments. It thus expands the experimental repertoire for dynamic, multigene interrogation in complex systems.