Identifying chemical lead matter by high-throughput screening (HTS) has been a common practice in early stage drug discovery. Evolution of small-molecule library composition to include more drug-like molecules with desirable physical chemical properties combined with improving assay technologies has vastly enhanced the capability of HTS. However, HTS campaigns can still be plagued by false positives arising from nonspecific inhibitors. The generation of assay-ready plates has permitted an incremental advancement to the speed and efficiency of HTS but has the potential to enhance the occurrence of nonspecific inhibitors. A subtle change in the order of reagent addition to the assay-ready plates can greatly alleviate false-positive inhibition. Our case studies with six different kinase and protease targets reveal that this type of inhibition affects targets regardless of enzyme class and is unpredictable based on protein construct or inhibitor chemical scaffold. These case studies support a model where a diversity set of compounds should be tested first for hit rates as a function of order of addition, carrier protein, and relevant mechanistic studies prior to launch of the HTS campaign.