Human mesenchymal stem cells are a good candidate to repair and to regenerate tissues for regenerative medicine applications. Their use in combination with 3D scaffolds has been largely studied in vitro to characterize their properties and differentiation potential prior to apply them in vivo. One of the most important clues in vitro is given by their proliferation trend, leading to information about their viability, their wellness, their interaction with scaffolds, etc. In order to measure the proliferation of hMSCs on scaffolds for regenerative medicine, it is important to adopt accurate counting methods in both research and diagnostic studies. This work aims to develop a reproducible method for hMSCs proliferation measurement in 3D cell cultures on coralline scaffolds (Biocoral®). Results demonstrated that: proliferation curves obtained in this work are reproducible at different initial cell densities on several scaffolds cultured with hMSC in long term experiments (3 weeks).