Purpose: The objective of this study was to establish a method for accurate cell counting from matrix-rich cell sheets in the clinical setting. Materials and Methods: Human periodontal ligament (HPDL) cells were obtained from healthy donors to prepare PDL cell sheets. To obtain single cell suspensions, the cell sheets were treated with three different enzymatic formulations: collagenase alone, trypsin-ethylenediaminetetraacetic acid (EDTA) alone, and a combination of collagenase and trypsin-EDTA. After cell dispersion, cell numbers and cell survival rates were measured. To evaluate damage to the cell surfaces from the enzymes, the dispersed cells were analyzed by a flow cytometer with an anti-alkaline phosphatase antibody. Results: Treatment with collagenase alone or trypsin-EDTA alone dispersed few cells from HPDL cell sheets. In contrast, combined treatment with collagenase and trypsin-EDTA successfully produced a large amount of single cells from cell sheets. Flow cytometry analysis showed that single cells obtained by combined use of collagenase and trypsin-EDTA preserved alkaline phosphatase epitopes on the cell surfaces. Conclusions: Cell sheets rich with extracellular matrix were dispersed via combined treatment with collagenase and trypsin-EDTA without destroying the expression of cell surface markers. The results suggest that this method would be useful for determining the accurate cell number of cell sheets for cell therapies and should also be applicable for other kinds of matrixrich cell sheets. Keywords: cell sheet, collagenase, extracellular matrix, human periodontal ligament cells, trypsin-ethylenediaminetetraacetic acid