Structural, bioanalytical, characterization, and quality control studies are critical for successful drug development. These studies must be as accurate, sensitive, and selective as possible, and liquid chromatography coupled to tandem mass spectrometry (LC–MS–MS) has been the technique of choice for many areas of small molecule analysis for the past 30 years. During that time, rapid improvements in analytical technologies have supported the development of more sensitive and robust methods. However, the pharma and biopharma industry continues to need more powerful instruments and more diverse methods, particularly as therapeutics have expanded to include large molecules. This work follows on from an earlier article that explored the limitations of LC–MS–MS for bioanalysis of biologics. This article considers some of the current issues for analysis of small and large molecules, and emerging trends in method development.