Antibody-drug conjugates (ADCs) are emerging as powerful tools in cancer therapy. Evaluating their drug disposition requires the development and validation of analytical methods to obtain accurate quantitative results, which depend on understanding the ADC structural properties and selecting appropriate analytical platforms. Liquid chromatography-mass spectrometry (LC-MS) is a key technology for ADC bioanalysis, enabling the quantification of payloads, linkers, total antibodies, ADCs, and drug-to-antibody ratio (DAR). This review highlights the strategies and challenges in developing analytical methods for quantifying ADC components in biological samples using LC-MS with a focus on their constituent units. In addition, it addresses the validation requirements of these quantitative analytical methods during drug development.