One of the main reasons for drug failures in clinical development, or postmarket launch, is lacking or compromised safety margins at therapeutic doses. Organ toxicity with poorly defined mechanisms and adverse drug reactions associated with on- and off-target effects are the major contributors to safety-related shortfalls of many clinical drug candidates. Therefore, to avoid high attrition rates in clinical trials, it is imperative to test compounds for potential adverse reactions during early drug discovery. Beyond a small number of targets associated with clinically acknowledged adverse drug reactions, there is little consensus on other targets that are important to consider at an early stage for in vitro safety pharmacology assessment. We consider here a limited number of safety-related targets, from different target families, which were selected as part of in vitro safety pharmacology profiling panels integrated in the drug-development process at Novartis. The best way to assess these targets, using a biochemical or a functional readout, is discussed. In particular, the importance of using cell-based profiling assays for the characterization of an agonist action at some GPCRs is highlighted. A careful design of in vitro safety pharmacology profiling panels allows better prediction of potential adverse effects of new chemical entities early in the drug-discovery process. This contributes to the selection of the best candidate for clinical development and, ultimately, should contribute to a decreased attrition rate.