Antibody-drug conjugates (ADCs) are monoclonal antibodies with covalently bound cytotoxic drugs. They are designed to target tumor antigens selectively and offer the hope of cancer treatment without the debilitating side-effects of conventional therapies. The concept of ADCs is not new; however, development of these therapeutics is challenging and only recently are promising clinical data emerging. These challenges include ADC bioanalysis, such as quantifying in serum/plasma for PK studies and strategies for assessing immunogenicity. ADCs have complex molecular structures incorporating large- and small-molecule characteristics and require diverse analytical methods, including ligand-binding assays and MS-based methods. ADCs are typically mixtures with a range of drug-to-antibody ratios. Biotransformations in vivo can lead to additional changes in drug-to-antibody ratios resulting in dynamically changing mixtures. Thus, a standard calibration curve consisting of the reference standard may not be appropriate for quantification of analytes in vivo and represents a unique challenge. This paper will share our perspective on why ADC bioanalysis is so complex and describe the strategies and rationale that we have used for ADCs, with highlights of original data from a variety of nonclinical and clinical case studies. Our strategy has involved novel protein structural characterization tools to help understand ADC biotransformations in vivo and use of the analyte knowledge gained to guide the development of quantitative bioanalytical assays.