The Mechanism of Action (MoA) of a drug is generally represented as a small, non-tissue-specific repertoire of high-affinity binding targets. Yet, drug activity and polypharmacology are increasingly associated with a broad range of off-target and tissue-specific effector proteins. To address this challenge, we have leveraged a microfluidics-based Plate-Seq technology to survey drug perturbational profiles representing >700 FDA-approved and experimental oncology drugs, in cell lines selected as high-fidelity models of 23 aggressive tumor subtypes. Built on this dataset, we implemented an efficient computational framework to define a tissue-specific protein activity landscape of these drugs and reported almost 50 million differential protein activities derived from drug perturbations vs. vehicle controls. These analyses revealed thousands of highly reproducible and novel, drug-mediated modulation of tissue-specific targets, leading to generation of a proteome-wide drug functional network, characterization of MoA-related drug clusters and off-target effects, dramatical expansion of druggable human proteome, and identification and experimental validation of novel, tissue-specific inhibitors of undruggable oncoproteins, most never reported before. The drug perturbation profile resource described here represents the first, large-scale, whole-genome-wide, RNA-Seq based dataset assembled to date, with the proposed framework, which is easily extended to elucidating the MoA of novel small-molecule libraries, facilitates mechanistic exploration of drug functions, supports systematic and quantitative approaches to precision oncology, and serves as a rich data foundation for drug discovery.