In this paper we report the use of the optical technique applied in the cylinder of an optically accessible engine equipped with the latest-generation diesel engine head of a European passenger car. The injection strategy with high percentage of EGR, characteristic of real engine operating point, was adopted. Alternative diesel fuels were used. In particular, rapeseed methyl ester (RME) and gas to liquid (GTL) were selected as representative of 1st and 2nd generation alternative diesel fuels, respectively. Combustion analysis was carried out in the engine combustion chamber by means of 2D spectroscopic measurements from UV to visible. These measurements helped to analyze the chemical and physical events occurring during the mixture preparation and the combustion development. Ultraviolet (UV) digital imaging was also performed and the presence of characteristic radical, like OH, in the various phases of combustion was detected as well. OH spatial distribution and temporal evolution were measured. Two color pyrometry technique was applied in order to measure the soot volume fraction within the combustion chamber. The GTL fuel showed better performance in terms of indicated mean effective pressure (IMEP) with respect to the diesel reference fuel with different effects on particulate matter (PM) and gaseous emissions. It showed the highest in cylinder soot production, while the OH radical had maximum intensity value close to the reference diesel (REF) one. On the other hand, the RME fuel showed a decrease in IMEP that can be adjusted with a little increase of fuel injected quantity, and very low production of soot in the cylinder and PM at the exhaust compared to the diesel reference fuel. Finally, the OH radical had the lowest intensity value.