Genetic interactions influence many phenotypes and can be used as a powerful experimental tool to discover functional relationships between genes. Here we describe a robust and scalable method to systematically map genetic interactions in human cancer cells using combinatorial RNAi and high-throughput imaging. Through automated, single-cell phenotyping, we measured genetic interactions across a broad spectrum of phenotypes, including cell count, cell eccentricity and nuclear area. We mapped genetic interactions of epigenetic regulators in colon cancer cells, recovering known protein complexes. Our study also revealed the prospects and challenges of studying genetic interactions in human cells using multiparametric phenotyping.