Bone growth is an essential part of skeletal development during childhood and puberty. Accurately characterizing longitudinal bone growth helps to better understand the determining factors of peak bone mass, which has impacts on bone quality later in life. Animal models were largely used to study longitudinal bone growth. However, the commonly used histology-based method is destructive and unable to follow up the growth curve of live animals in longitudinal experiments. In this study, we validated an in vivo micro-CT-based method against the histology-based method to quantify longitudinal bone growth rates of young rats non-destructively. CD (Sprague Dawley) IGS rats aged 35, 49 and 63 days received the same treatments:​ two series of repeated in vivo micro-CT scans on their proximal hind limb at a five-day interval, and two calcein injections separated by three days. The longitudinal bone growth rate was quantified by registering time-lapse micro-CT images in 3D, calculating the growth distance on registered images, and dividing the distance by the five-day gap. The growth rate was also evaluated by measuring the 2D distance between consecutive calcein fluorescent bands on microscopic images, divided by the three-day gap. The two methods were both validated independently with reproducible repeated measurements, where the micro-CT-based method showed higher precision. They were also validated against each other with low relative errors and a strong Pearson sample correlation coefficient (0.998), showing a significant (p < 0.0001) linear correlation between paired results. We conclude that the micro-CT-based method can serve as an alternative to the histology-based method for the quantification of longitudinal growth. Thanks to its non-invasive nature and true 3D capability, the micro-CT-based method helps to accommodate in vivo longitudinal animal studies with highly reproducible measurements.