The ability of hepatocytes to enter the cell cycle and regenerate the liver after tissue loss provides an in vivo model to study the regulation of proliferation and organ regeneration. The extent of hepatocyte proliferation is directly proportional to the amount of resected liver tissue, and 2/3 partial hepatectomy (2/3 PH) leads to highly synchronized hepatocyte cell-cycle entry and progression. This surgical technique was first described in rats and requires modification for application in mice. Lack of standardization of 2/3 PH in mice has caused discrepancies in the results obtained in different laboratories. Here, we provide a protocol and a movie describing a straightforward surgical technique, which takes 15–20 min, to consistently remove two-thirds of the liver in mice. As this protocol is not associated with mortality and gives highly reproducible results, we hope that it will be widely used and serve to standardize 2/3 PH in mice.