New imaging techniques using near-infrared (NIR) femtosecond lasers (fs-lasers) in multiphoton laser scanning microscopy (MPLSM) have great potential for in vivo applications, particularly in human skin. However, little is known about possible risks. In order to evaluate the risk, a “biological dosimeter” was used. We irradiated fresh human skin samples with both an fs-laser and a solar simulator UV source (SSU). DNA damage introduced in the epidermis was evaluated using fluorescent antibodies against cyclobutane-pyrimidin-dimers (CPDs) in combination with immunofluorescence image analysis. Four fs-irradiation regimes (at wavelength) were evaluated differing in laser power and step width of horizontal scans. Fs-irradiation did not give CPDs at or irradiation power using 10 horizontal scans every . CPDs could be seen at laser power and step size and at using step width. Quantitative comparison of SSU-induced CPDs showed that the laser irradiation regime is comparable to UV-irradiation, giving 0.6 minimal erythemal dose (MED). The irradiation regime was comparable to 0.45 MED. Under these experimental conditions, the risk of DNA damage due to fs-laser irradiation on skin is in the range of natural UV-exposure.