The auto-ignition process plays a major role in the combustion, performance, fuel economy, and emission in diesel engines. The auto-ignition quality of different fuels has been rated by its cetane number (CN) determined in the cooperative fuel research engine, according to ASTM D613. More recently, the ignition quality tester (IQT), a constant volume vessel, has been used to determine the derived cetane number (DCN) to avoid the elaborate, time consuming, and costly engine tests, according to ASTM D6890. The ignition delay (ID) period in these two standard tests and many investigations has been considered to be the time period between start of injection (SOI) and start of combustion (SOC). The ID values determined in different investigations can vary due to differences in instrumentation and definitions. This paper examines the different definitions and the parameters that effect ID period. In addition, the activation energy dependence on the ID definition is investigated. Furthermore, results of an experimental investigation in a single-cylinder research diesel engine will be presented, while the charge density is kept constant during the ID period. The global activation energy is determined and its sensitivity to the charge temperature is examined.