Increasingly targeted in drug discovery, protein-protein interactions challenge current high throughput screening technologies in the pharmaceutical industry. Developing an effective and efficient method for screening small molecules or compounds is critical to accelerate the discovery of ligands for enzymes, receptors, and other pharmaceutical targets. Here, we report developments of methods to increase the signal-to-noise ratio for screening protein-protein interactions using atomic force microscopy (AFM) force spectroscopy. We have demonstrated the effectiveness of these developments on detecting the binding process between focal adhesion kinases with protein kinase B (Akt1), which is a target for potential cancer drugs. These developments include optimized probe and substrate functionalization processes and redesigned probe-substrate contact regimes. Furthermore, a statistical-based data processing method was developed to enhance the contrast of the experimental data. Collectively, these results demonstrate the potential of the AFM force spectroscopy in automating drug screening with high throughput.