Distraction　osteogenesis　(DO)　is　widely　utilized　for　treating　limb　length　discrepancy,　nonunion,　bone　deformities　and　defects.　This　study　sought　to　develop　a　4D　time-lapse　morphometry　method　to　quantify　bone　formation　and　resorption　in　mouse　femur　during　DO　based　on　image　registration　of　longitudinal　in　vivo　micro-CT　scans.　Female　C57BL/6　mice　(n　=　7)　underwent　osteotomy,　followed　by　5　days　of　latency,　10　days　of　distraction　and　35　days　of　consolidation.　The　mice　were　scanned　with　micro-CT　at　Days　5,　15,　25,　35,　45,　and　50.　Histological　sectioning　and　Movat　Pentachrome　straining　were　performed　at　Day　50.　After　registration　of　two　consecutive　micro-CT　images　of　the　same　bone　(day　x　and　day　y),　the　spatially-　and　temporally-linked　sequences　of　formation,　resorption　and　quiescent　bones　at　the　distraction　gap　were　identified　and　bone　formation　and　resorption　rates　(BFRdayx-y　and　BRRdayx-y)　were　calculated.　The　overall　percentage　error　of　the　registration　method　was　2.98%　±　0.89%　and　there　was　a　strong　correlation　between　histologically-measured　bone　area　fraction　and　micro-CT-determined　bone　volume　fraction　at　Day　50　(r　=　0.89,　p　＜　0.05).　The　4D　time-lapse　morphometry　indicated　a　rapid　bone　formation　during　the　first　10　days　of　the　consolidation　phase　(BFRday15–25　=　0.14　±　0.05　mm3/day),　followed　by　callus　reshaping　via　equivalent　bone　formation　and　resorption　rates.　The　4D　time-lapse　morphometry　method　developed　in　this　study　allows　for　a　continuous　quantitative　monitoring　of　the　dynamic　process　of　bone　formation　and　resorption　following　distraction,　which　may　offer　a　better　understanding　of　the　mechanism　for　mechano-regulated　bone　regeneration　and　aid　for　development　of　new　treatment　strategies　of　DO.　©　2024　Orthopaedic　Research　Society.