DiscoveRx's PathHunter® platform is a simple, cell-based, chemiluminescent assay format that allows for screening large numbers of small molecules or hybridoma supernatants in a one-step, no-wash format making it both user-friendly and HTS-compatible. In this study, we demonstrate the 3456- and 1536-well application of DiscoveRx's PathHunter GPCR β-Arrestin assays as well as PathHunter® Receptor Tyrosine Kinase assays using the Echo® liquid handler (Labcyte Inc.) for acoustic, non-contact compound and assay reagent dispensing to AURORA high performance 3456 plates. All assays are analyzed on the new highly sensitive BMG LABTECH PHERAstar FS microplate reader. Precise liquid handling and ultrasensitive instrumentation further enhances the speed of processing allowing a complete 3456-well PathHunter® assay plate to be read in less than 3 minutes. Robustness of the PathHunter assay is further enhanced on a BMG LABTECH instrument and in combination with the Echo liquid handler, it reduces assay variability, improves Z’ values as well as data quality. Performing a cell-based assay at <5 µL volume translates to very low compound requirement, fewer cells and low reagent usage. A highly miniaturized PathHunter assay in combination with the BMG LABTECH PHERAstar FS and the Labcyte Echo liquid handler provides pharma/biotech/academic consortia a faster, simpler, cost-effective and a more efficient way to screen large compound libraries thereby enabling cost effective drug discovery campaigns.