Site-specific immobilization of proteins and peptides on a sensor surface represents a significant challenge for bioanalytical applications such as surface plasmon resonance (SPR). The most common protocols for covalent protein immobilization usually result in heterogeneous presentation of the ligand at the surface, which can in some instances yield conflicting results with analogous data obtained in solution. Here, we discuss two complementary and generic bioconjugation methods that allow chemoselective immobilization of peptides and proteins via either their C-terminus (native chemical ligation) or their N-terminus (oxime ligation). While the protocols described in this chapter were designed for use in a Biacore instrument, the methods should also be applicable to other SPR instruments and, with slight adjustments, to many other types of bioanalytical applications that rely on protein-functionalized surfaces.