The JAK/STAT1 signaling pathway is known to be activated by type I/II interferons such as interferon-gamma and interferon-alpha. In this pathway, binding of these cytokines to their respective cell-surface receptors results in the activation of JAKs, which in turn phosphoactivate STAT1 proteins at a specific tyrosine residue (Y701). LanthaScreen® STAT1 U2OS is a human cell line that constitutively expresses a GFPSTAT1 fusion protein. The JAK/STAT1 signaling pathway is known to be functionally intact in this cell line; therefore, the GFP-STAT1 fusion protein serves as a substrate for the IFN-gamma-inducible phosphorylation by JAKs. Using this cell line, a homogeneous immunoassay has been developed in which the phosphorylation state of GFP-STAT1 is detected in cell lysates using a terbium-labeled anti-pY701-STAT1 antibody in a time-resolved FRET (TR-FRET) readout. The GFP-STAT1 DNA expression construct was transfected into U2OS cells using Lipofectamine™ LTX Reagent, followed by selection with blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker. Using the lytic TR-FRET immunoassay, this cell line is validated for EC50 and Zfi-factors under optimized conditions using IFN-gamma as a ligand for JAK-mediated GFP-STAT1 phosphorylation. This assay has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance, and assay development time. Additional information using alternate stimuli and alternate assay protocols is available.