Detailed specificity analysis is critical for drug development, as even minimal off-target binding may cause serious adverse events. Off-target binding is the primary cause of preclinical and early-stage clinical trial failures. Recent clinical data and our own profiling indicate that biotherapeutics frequently (~25%) display polyspecificity, resulting in failed clinical trials and severe adverse events in patients. As a result, specificity profiling is an important regulatory requirement for biotherapeutic categories, such as monoclonal antibodies and CAR T cell therapy. The primary methods for profiling the specificity of biotherapeutics include tissue cross-reactivity studies and cell-based expression arrays of structurally intact membrane proteins (“cell expression arrays”). This chapter details the advantages of each approach and discusses how to interpret outcomes when off-target interactions are discovered. More thorough specificity profiling of lead therapeutics will improve off-target binding prediction and minimize risk in biotherapeutic discovery programs prior to clinical development.