Surface plasmon resonance (SPR) biosensors represent one of the most advanced sensing technologies for real-time studies of biomolecular interactions. In this thesis, methods for functionalization of SPR substrates were optimized and studied via spectroscopic methods. Effects related to the SPR sensor microfluidic interface on the measured biomolecular interactions were analyzed, and furthermore, means to decrease mass-transport limitations were proposed. Several SPR-based assays regarding the detection of nucleic acids were developed, which allow for the detection of physiologically relevant concentrations of nucleic acids as well as point mutations in a nucleic acid sequence. Assays for the determination of the enzymatic activity of HIV integrase and ribonuclease H were developed. These assays can be employed for the design and synthesis of molecules that function either as antiviral drugs or as gene-regulating agents.