Hepatocellular carcinoma (HCC) may metastasize to many organs. The survival rate is almost zero for metastatic HCC patients. Molecular mechanisms of HCC metastasis need to be understood better and new therapies must be developed. We have developed the “in vivo microscopy” to study the mechanisms that govern liver tumor cells spreading through the microenvironment in vivo. A recently developed “in vivo flow cytometer” combined with real-time confocal fluorescence imaging is used to assess spreading and the circulation kinetics of liver tumor cells. We measure the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which are from the same origin and obtained by repetitive screenings in mice. More than 60% of the HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells possess noticeably slower depletion kinetics. In comparison, less than 40% of the HepG2 cells are depleted within the first hour. The differences in depletion kinetics might provide insights into early metastasis processes. The mechanisms of HCC metastasis need to be understood better and new therapies must be developed. We have developed the “in vivo microscopy” to study the mechanisms that govern liver tumor cells spreading through the microenvironment in vivo. A recently developed “in vivo flow cytometer ” combined with real-time confocal fluorescence imaging is used to assess spreading and the circulation kinetics of liver tumor cells. measure the depletion kinetics of two related human HCC cell lines, high-metastatic HCCLM3 cells and low-metastatic HepG2 cells, which are from the same origin and obtained by repetitive screenings in mice. More than 60% of the HCCLM3 cells are depleted within the first hour. Interestingly, the low-metastatic HepG2 cells have a noticeably slower depletion kinetics. In less than 40% of the HepG2 cells are depleted within th e first hour. The differences in depletion kinetics might provide insights into early metastasis processes.