Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture. However, the systematic errors in the application of western blotting analysis are frequently to be found, which may compromise the interpretation of results. To make a valid application of western blotting, it is essential to begin with three independent biological replicates. Subsequently, a more reliable normalization method is in urgent need for western blotting analysis and using reference proteins is the currently preferred method of normalization. Additionally, identification of valid reference proteins is crucial for western blotting analysis and it should be examined carefully in relation to the cell or tissue types when using housekeeping proteins as internal standards.