The light-induced fluorescence (LIF) represents an important tool for the continuous improvement and further development of combustion engine systems regarding efficiency increase. In this work, the interactions between light-emitting diode (LED) and laser diode light radiation, engine oil/fuel and three fluorescence tracers are investigated on an application-related investigation system for the design of qualitative LIF experiments. Thereby two efficient light sources for engine combustion chamber lighting are presented. For different engine oil/fuel tracer combinations, the fluorescence is examined in its intensity and its spectrum depending on the temperature, concentration and temperature history. With oil temperature variations of up to 150 °C, changes in the fluorescence spectrum and fluorescence intensities that decrease by up to 80% are evident. For specific tracer oil/fuel mixtures, concentration-dependent maximum intensities and different temperature history behaviors can be revealed. The results shown support the design of spectral engine examination setups and give tracer dosage recommendations. Additionally, an engine LIF setup for in-cylinder tracking of engine operating media is presented.