During the endochondral ossification, the bone is formed from the cartilage. In this study, we fabricated scaffold-free spheroids using mouse pre-chondrocyte ATDC5 cells. Real-time PCR results represented that the spheroid culture for ATDC5 cells after 14 days up-regulated chondrocyte marker as well as hypertrophic chondrocyte marker compared beyond the 2-day cultured spheroids. In addition to the biochemical analysis, we also conducted 3D image analysis in conjunction with optical clearing techniques and confirmed the enlargement of the cells in the spheroid after the long-term culture. Our present study suggested that the spheroid culture for ATDC5 cells provoked the initial stage of endochondral ossification process involving the hypertrophy chondrocyte differentiation.