We investigated a viscous protein layer formed on self-assembled monolayers (SAMs) in crowded biological environments. The results were obtained through force spectroscopic measurements using colloidal probes and substantiated by exhaustive analysis using a quartz crystal microbalance with an energy dissipation technique. A hydrophobic SAM of n-octanethiol (C8 SAM) in bovine serum albumin (BSA) solution is buried under an adlayer of denatured BSA molecules and an additional viscous interphase layer that is five times more viscous than the bulk solution. C8 SAMs in fetal bovine serum induced a formation of a thicker adsorbed protein layer but with no observable viscous interphase layer. These findings show that a fouling surface is essentially inaccessible to any approaching molecules and thus has a new biological and physical identity arising from its surrounding protein layers. In contrast, the SAMs composed of sulfobetaine-terminated alkanethiol proved to be sufficiently protein-resistant and bio-inert even under crowded conditions due to a protective barrier of its interfacial water, which has implications in the accurate targeting of artificial particles for drug delivery and similar applications by screening any non-specific interactions. Finally, our strategies provide a platform for the straightforward yet effectual in vitro characterization of diverse types of surfaces in the context of targeted interactions in crowded biological environments.