The accurate determination of events at the interface between a biological system and nanomaterials is necessary for efficacy and safety evaluation of novel nano-enabled medical products. Investigating the interaction of proteins with nanoparticles (NPs) and the formation of protein corona on nanosurfaces is particularly challenging from the methodological point of view due to the multiparametric complexity of such interactions. This study demonstrated the application of localized surface plasmon resonance (LSPR) spectroscopy as a low-cost and rapid biosensing technique that can be used in parallel with other sophisticated methods to monitor nano-bio interplay. Interaction of citrate-coated gold NPs (AuNPs) with human plasma proteins was selected as a case study to evaluate the applicability and value of scientific data acquired by LSPR as compared to fluorescence spec roscopy, which is one of the most used techniques to study NP interaction with biomolecules. LSPR results obtained for interaction of AuNPs with bovine serum albumin, glycosylated human transferrin, and non-glycosylated recombinant human transferrin correlated nicely with the adsorption constants obtained by fluorescence spectroscopy. This ability, complemented by its fast operation and reliability, makes the LSPR methodology an attractive option for the investigation of a nano-bio interface.