High throughput screening (HTS) has become a pivotal part of drug discovery for identifying potential lead molecules for specific disease targets. The success of screening is measured by several factors including the throughput and cost of the assays, but ultimately by the number of HTS hits that progress forward through the drug discovery pipeline. Miniaturization of assays by conversion from 96- to 384- to 1536- wells to well-less platforms, and the utilization of automated equipments are methodologies implemented to increase the throughput and reduce the cost of the screen. Pooling of test compounds is also an effective, though controversial method to enhance screening throughput while reducing reagent costs. Herein, we describe specific measures implemented at Abbott Laboratories to enhance the efficiency of 10- mixture orthogonal screening, and the strategy developed to reduce the rate of false positives and eliminate false negatives. Results from a comparison study of mixtures versus single compound screening will be discussed for three distinct screening formats: a GPCR functional assay, a GPCR binding assay and a tyrosine kinase assay.