Western blot is one of the most reliable methods to characterize protein expression. Using straightforward protocols and relatively inexpensive reagents, it provides a semi-quantitative visual assessment of even low-abundance targets. This is achieved through partial protein purification according to size, prior to highly specific antibody-based detection of the target of interest. Western blot has been improved with innovative detection methods, more sensitive imaging technologies, and a variety of automated instrumentation. More recently it has evolved into a sophisticated gel-free, blot-free technique. With many researchers generating western blot data to support experimental results, and most antibody manufacturers using western blot to validate their products, this trusted method remains extremely popular. Understanding the different options, how they work, and the benefits they offer can increase the chances of western blot success.