Western blotting is a widely used technique for identifying the expression of a particular protein. This chapter presents a comprehensive methodology for characterizing transgenic plants in Arabidopsis thaliana using Western blotting. Western blotting allows for the accurate evaluation of protein expression levels, which is essential for confirming the integration and functionality of transgenes. The technique consists of several stages, starting with the preparation of plant samples and protein extraction. Then extracted proteins are subjected to electrophoretic separation on polyacrylamide gels and then transferred onto a membrane. Subsequently, the membrane is examined using specialized antibodies that specifically bind to the transgenic product, enabling the identification and measurement of the produced protein. Important factors such as the specificity of antibodies, the stability of proteins, and the use of loading controls are taken into account to guarantee the precision and consistency of the outcomes. This technique provides researchers with a systematic approach to efficiently analyze transgenic plants.