Biochemical assays are valuable for discovery and evaluation of compounds that specifically modulate activity of therapeutic targets outside of the context of a living cell. Key feature of biochemical assays is their inherent low biological complexity which makes them robust and reliable. We prefer and recommend ratiometric biochemical assay formats that quantitatively measure specific on-target activities. Ratiometric assays can eliminate signal variation and errors caused by non-specific interference of the test compound. The use of red-shifted dyes or time-resolved fluorescence read-outs can further help minimizing issues introduced by auto-fluorescence of test compounds.

Cell-based assays measure activity of drug compounds on pharmaceutical targets via a relevant read-out in the context of a living cell. Cell-based assays are required for drug targets, which are difficult to purify or non-functional outside of the context of a living cell. Examples include the large family of G protein-coupled receptors (GPCRs), as well as ion channels and transporters located to cellular membranes, or other target proteins composed of multiple subunits. Despite their pronounced biological complexity, cell-based assays allow measurement of convoluted signaling pathways and assessment of additional properties of test compounds including efficacy, cytotoxicity, and other effects not directly related to target-binding. Cell-based assays that intercept activity signals close to the target are usually less prone to signal interference compared to readouts measured further downstream of the target. However, these distally measured readouts are often preferred due to their enhanced sensitivity and ease of use. If available, we use reference compounds to calibrate a cell-based assay, and confirm that all measured cellular responses are specific to the target of interest. Additionally, "High content", multi-parametric live-cell microscopy readouts and Artificial Intelligence (AI)-guided approaches such as cell painting have further potential to effectively distinguish target-related activities from non-specific "off-target" compound effects.