The manipulation of cells by femtosecond (fs) laser pulses became a very important tool in cell biology. In terms of learning more about the function of the cell compartments and the cell dynamics, single cell organelles are manipulated by laser pulses. Meanwhile the cell reaction is observed by different microscopy methods. The parameters of the laser irradiation have to be chosen carefully to minimize unwanted side effects during the treatment and to prevent cell damage or cell death. In many applications, it is not known what happens due to the laser irradiation on the molecular level. The formation of reactive oxygen species (ROS) is an often predicted effect due to photo disruption in biologic tissue. In this paper, we present our study of the ROS formation during the irradiation of fs laser pulses for disruption of single cell organelles. The quantity of ROS formation depends strongly on the pulse energy of the laser. Therefore the creation of ROS was additionally studied while scanning the laser at low energy for multiphoton microscopy.