High-affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (KD) of the molecular pair, with avidin:biotin being the state-of-the-art molecular pair (KD ~ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high-affinity protein pair, barstar:barnase (KD ~ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non-negligible background due to the non-specific binding. A quantitative assessment of the non-specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin-based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non-specific binding, showing good prospects for high-sensitivity rare biomolecular event nanoproteomic assays.