The Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay provides a fast, sensitive, non-toxic and non-radioactive method for the detection of nascent protein synthesis utilizing fluorescence microscopy and high-content imaging. Click-iT® AHA is an amino acid analog of methionine containing an azido moiety. Similar to 35S-methionine, Click-iT® AHA is fed to cultured cells and incorporated into proteins during active protein synthesis. Detection of the incorporated amine acid utilizes a chemoselectiove ligation or “click" reaction between an azide and an alkyne, where the azido modified protein is detected with the green-fluorescent Alexa Fluor® 488 alkyne. The Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay has been successfully tested in A549 and U-2 OS cells with a variety of reagents that inhibit protein synthesis including cycloheximide, anisomycin and puromycin in both dose response and Min⁄Max format.