MSCs (mesenchymal stem cells) are planned foruse in regenerative medicine to offset age-dependent alterations. However, MSCs are affected by replicative senescence associated with decreasing proliferation potential, telomere shortening and DNA damage during in vitro propagation. To monitor in vitro senescence, we have assessed the integrity of DNA by the alkaline comet assay. For optimization of the comet assay we have enhanced the stability of comet slides in liquid and minimized the background noise of the method by improving adhesion of agarose gels on the comet slides and concentrating cells on a defined small area on the slides. The modifications of the slide preparation increase the overall efficiency and reproducibility of the comet assay and minimize the image capture and storage. DNA damage of human MSCs during in vitro cultivation increased with time, as assessed by the comet assay, which therefore offers a fast and easy screening tool in future efforts to minimize replicative senescence of MSCs in vitro.