Abstract: Objective To assess the value of two-color infrared fluorescence imaging system in detecting protein phosphory-lation in comparison with chemiluminescent detection. Method The lung tissue homogenate of mice treated with lipopolysac-charide (LPS) for different time lengths were prepared to separate the proteins by SDS-polyacrylamide gel electrophoresis followed by transfer of the proteins onto PVDF membranes. The membranes were incubated with the antibodies against total p42/44 MAPK/phospho-p42/44 MAPK, and then with goat anti-mouse or anti-rabbit secondary antibodies conjugated to Alexa Fluor 680, IRDye 800 or horseradish peroxidase. The blotted proteins were detected and quantified using Odyssey infrared imaging system or chemiluminescent detection. Results LPS treatment rapidly induced p42/44 MAPK phosphorylation, which reached the peak level 1 h after the treatment and resumed the baseline level at 12 h. Consistent results were obtained by the two detection methods, but two-color infrared fluorescence imaging system showed better sensitivity in detecting the target protein, and was easy to manipulate with good efficiency and capable of analyzing two proteins simultaneously. Conclusion Two-color infrared fluorescence system is a powerful system for detecting phosphorylation of proteins.