Drug action can be rationalized as interaction of a molecule with proteins in a regulatory network of targets from a specific biological system. Both drug and side effects are often governed by interaction of the drug molecule with many, often unrelated, targets. Accordingly, arrays of protein-ligand interaction data from numerous in vitro profiling assays today provide growing evidence of polypharmacological drug interactions, even for marketed drugs. In vitro off-target profiling has therefore become an important tool in early drug discovery to learn about potential off-target liabilities, which are sometimes beneficial, but more often safety relevant. The rapidly developing field of in silico profiling approaches is complementing in vitro profiling. These approaches capitalize from large amounts of biochemical data from multiple sources to be exploited for optimizing undesirable side effects in pharmaceutical research. Therefore, current in silico profiling models are nowadays perceived as valuable tools in drug discovery, and promise a platform to support optimally informed decisions.