Protein resistant or “non-fouling” surfaces are of great interest for a variety of biomedical and biotechnology applications. This article briefly reviews the development of protein resistant surfaces, followed by recent research on a new methodology to fabricate non-fouling surfaces by surface-initiated polymerization. We show that polymer brushes synthesized by surface-initiated polymerization that present short oligo(ethylene glycol) side chains are exceptionally resistant to protein adsorption and cell adhesion. The importance of the protein and cell resistance conferred by these polymer brushes is illustrated by their use as substrates for the fabrication of antibody microarrays that exhibit femtomolar limits of detection in complex fluids such as serum and blood with relaxed requirements for intermediate wash steps. This example highlights the important point that the reduction in background noise afforded by protein-resistant surfaces can greatly simplify the development of ultrasensitive heterogeneous, surface-based clinical and proteomic assays with increased sensitivity and utility.