Assays, both high-throughput and lower throughput, are an integral part of the drug discovery process and are used for identifying and validating potential drug candidates. Many biochemical and cell-based assays utilize fluorescence and to a lesser extent absorbance readouts so it is important to consider the impact on the assay quality of a given detection method. There are many fluorophores available that span a wide energy spectrum, and it is important to select the appropriate fluorophore and assay conditions for a given assay to minimize assay interference. Additionally, many of the small molecules found in libraries used for screening are themselves fluorescent, leading to potential false positives through interference. Similarly, for absorbance assays, colored compounds can interfere with the detection method depending on the concentration and extinction coefficient. Assays can be designed to minimize the interference from the library itself, and counterassays can be utilized to identify potential compounds that interfere with the detection method. Orthogonal assays using a different detection method can also be employed to further validate HTS hits. It is important to weed out assay artifacts as early as possible in the drug discovery process to avoid spending time and resources pursuing compounds that are not actually impacting the desired biology but rather are false positives.