The energy gap of molecular semiconductors is a critical parameter in molecule-based devices because it fundamentally determines the operating principle and device performance, such as the charge transfer driving force. Hence, an accurate quantification of the energy gap of a molecular semiconductor is essential. The hot electron technique, which is implemented using a hot electron transistor and ballistic electron emission microscope, has been verified as the most powerful characterization in recent years. By monitoring the charge transport on the metal/molecule interface, an electron-injection barrier (or LUMO) and hole-injection barrier (or HOMO) can be achieved, which contributes to the electronic energy gap determination. In this review, a comprehensive comparison of these two techniques was made. It helps us to select a suitable method in specific situations and provides a profound comprehension of the charge transport process in molecular semiconductor and molecule-based devices.