In the era of proteomics, high-throughput screening of posttranslational modification states of proteins, especially protein phosphorylation, is considered of utmost importance. However, current protein phosphorylation detection methods depend on either the combination of proteolysis and mass spectrometry, or time-consuming immunoassay that requires inevitable washing processes. As a way to rapidly assay protein phosphorylation events, here we propose the use of Open Sandwich immunoassay that detects antigen- dependent stabilization of antibody variable region (Fv). As a model system, the heavy and light chain variable regions (V-H/V-L) of anti-phosphotyrosine antibody PY20 were used to evaluate its performance. When V-H/V-L interaction was first estimated by phage ELISA, wild-type Fv showed a modest phosphotyrosine (PY)-dependent increase in signal. However, after screening of mutants at an interface residue, one with weak VH/VL interaction (HQ39R) showed markedly improved (> 200%) antigen- dependent signals. Using this mutant, two fusion proteins in which each variable region fragment was tethered to a GFP color variant were made (V-H-eYFP/V-L-eCFP) to monitor PY-induced fluorescence resonance energy transfer (FRET) between them. The results showed significant PY- or tyrosine phosphorylated peptide-induced enhancement in FRET in homogeneous solutions, indicating applicability of the method for rapid screening of tyrosine phosphorylation in vitro or in situ and possibly in vivo.