Analytical ultracentrifugation (AUC) is a first principle biophysical technique which has been used for over a century to investigate the behaviour of biomolecules. AUC encompasses a range of experimental approaches which enable the investigation of the size, shape, and interactions of macromolecules in solution. In contrast to alternative methods of characterisation, AUC data reveals fundamental physical characteristics of the molecule being investigated. While the technique is well established within the annals of biochemistry, improvements to both the physical instrumentation and the data processing software have provided new opportunities for the use of AUC. Some of the key aspects of experimental design as well as several examples of the modern applications of AUC will be presented to encourage its use.