At any time in vitro or in vivo expressed unlabeled proteins have to be quantified it is difficult to find a reliable method, especially with nonpurified samples. Quantification via protein activity can result in too low levels if the proteins analyzed tend to aggregate into inactive forms. Here, wild-type green fluorescent protein (GFPwt) was expressed in high amounts in vitro using the Rapid Translation System 500 based on Escherichia coil lysates. Fluorescent activity was determined in dependence of oxygen and compared to total protein levels. In the presence of low amounts of oxygen only 16% of the whole GFPwt amounts were detectable via determination of fluorescence activity. A reliable method to easily quantify whole protein levels even without specific antibodies and without purification steps by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining is described.