Replicative senescence has major impact on function and integrity of cell preparations. DNA-methylation (DNAm) changes are continuously acquired at specific CpG dinucleotides during in vitro culture. Such modifications can be used to estimate the state of cellular senescence for quality control of cell preparations. It is however unclear how senescence-associated DNAm changes are regulated and if they occur simultaneously throughout cell populations. In this study, we analyzed global DNAm profiles of human mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVEC) to demonstrate that senescence-associated DNAm changes are overall similar in these different cell types. Subsequently, an Epigenetic-Senescence-Signature, based on six CpGs, was either analyzed by pyrosequencing or by barcoded bisulfite amplicon sequencing. There was a good correlation between predicted and real passage numbers in bulk populations of MSCs (R² = 0.67) and HUVECs (R² = 0.97). Overall design: Bisulphite converted DNA of early (P4) and late passages (P20,P18,P13) from 3 donors was hybridised to the Illumina Infinium 450k Human Methylation Beadchip