The nonclinical development plan (NDP) for biotechnology-derived pharmaceuticals (biologics) as addressed in this review comprises aspects of nonclinical drug safety (toxicology and safety pharmacology), nonclinical pharmacokinetics (PKs) and nonclinical and clinical bioanalytics. A variety of bioanalytical methods are needed to describe PK behavior and to monitor immune response in laboratory animals and humans. The NDP will often differ from established programs for small molecules to account for target differences, a lack of or limited species crossreactivity and/or the limited applicability of long-term studies, as well as immunogenicity assessment. Special study designs, scientifically justified and driven by a case-by-case approach, might overcome these hurdles.