In a method of manufacturing a thin film transistor substrate, a first metal layer is formed on a first surface of a base substrate. The base substrate is cooled by contacting the first metal layer with a first cooling plate and by contacting a second surface of the base substrate with a second cooling plate. The first and second surfaces of the base substrate face opposite directions. A gate electrode is formed by patterning the first metal layer. A source electrode and a drain electrode are formed. The source electrode is spaced apart from the drain electrode. The source and drain electrodes partially overlap the gate electrode. A pixel electrode electrically connected to the drain electrode is formed.