Protein quantification is an integral part of many biological, biotechnological and medical activities, including process development, drug testing and diagnostics. The challenge of quantification is to determine the concentration of a specific target, which is often present in a crude mixture of many proteins and other molecules, such as nucleic acids, polysaccharides and metabolites. Well-known methods such as the enzyme-linked immunosorbent assay (ELISA) are accepted by the scientific community and regulatory authorities, but suffer inherent variability, may require expensive consumables, and depend strongly on operator experience, which can increase the variability of results by >10%. Surface plasmon resonance (SPR) spectroscopy is a suitable alternative in some cases because the limit of quantification can be as low as 10 μg L−1, with a coefficient of variation below 2%. In this chapter, we first introduce the principles of SPR and then highlight typical applications. We provide a detailed description of the assay steps and conclude with troubleshooting hints and recommended situations in which SPR provides superior results compared to traditional quantification methods such as ELISAs and other immunoassays.