Visualization of single-hole nozzles into quiescent ambient has been used extensively in the literature to characterize spray mixing and combustion. However in-cylinder flow may have some meaningful impact on the spray evolution. In the present work, visualization of direct diesel injection spray under both non-reacting and reacting operating conditions was conducted in an optically accessible two-stroke engine equipped with a single-hole injector. Two different high-speed imaging techniques, Schlieren and UV-Light Absorption, were applied here to quantify vapor penetration for non-reacting spray. Meanwhile, Mie-scattering was used to measure the liquid length. As for reacting conditions, Schlieren and OH* chemiluminescence were simultaneously applied to obtain the spray tip penetration and flame lift-off length under the same TDC density and temperature. Additionally, PIV was used to characterize in-cylinder flow motion. Results were compared with those from the Engine Combustion Network database obtained under quiescent ambient conditions in a high pressure high temperature vessel. Because of the air flow induced by piston movement, in-cylinder conditions in the two-stroke engine during the spray injection are highly unsteady, which has a significant impact on the spray development and interference on the spray visualization. From the comparison with quiescent data from the Engine Combustion Network, air flow induced by piston movement was found to slow down tip penetration. Moreover, both ignition delay and lift-off length under unsteady flow conditions show less sensitivity with ambient temperature than that of quasi-steady conditions.