A multinational pharmaceutical and biotechnology company survey was conducted to gain a better understanding of the use and value of the tissue cross-reactivity (TCR) assay in the development of biotherapeutic molecules. The majority of the molecules did not use TCR data as the only basis for determining species selection for toxicity studies (73%). For 95% of the molecules, the TCR data had no impact on the development strategy. For 2% of the molecules (1/56), TCR data was the sole source of information indicating a potential risk to patients. Unexpected or off-target binding was seen with 35% of the molecules, with the majority of this binding occurring in the CNS and reproductive organs. Tissues that were known or presumed to contain the target stained positively in 22% and 10% of molecules tested in non-human primate and human tissues, respectively. Tissues that were known or presumed to lack the target were negative for staining in 39% and 50% of molecules for non-human primate and human tissue, respectively. For 5% (6/110) of all the molecules, companies stated that toxicities would have been missed in animal studies or the clinic (i.e., not identified by clinical signs, histopathology, etc.) if the TCR studies had not been performed.