Drug resistance is a challenge in anticancer therapy, particularly with targeted therapeutics and cytotoxic compounds. In many cases, cancers can be resistant to the drug prior to exposure, i.e., possess intrinsic drug resistance. However, we lack target-independent methods to anticipate resistance in cancer cell lines or characterize intrinsic drug resistance without a priori knowledge of its cause. We hypothesized that cell morphology could provide an unbiased readout of drug sensitivity prior to treatment. We therefore isolated clonal cell lines that were either sensitive or resistant to bortezomib, a well-characterized proteasome inhibitor and anticancer drug to which many cancer cells possess intrinsic resistance. We then measured high-dimensional single-cell morphology profiles using Cell Painting, a high-content microscopy assay. Our imaging- and computation-based profiling pipeline identified morphological features typically different between resistant and sensitive clones. These features were compiled to generate a morphological signature of bortezomib resistance, which correctly predicted the bortezomib treatment response in seven of ten cell lines not included in the training dataset. This signature of resistance was specific to bortezomib over other drugs targeting the ubiquitin-proteasome system. Our results provide evidence that intrinsic morphological features of drug resistance exist and establish a framework for their identification.