Autofluorescence of the bone extracellular matrix (ECM) has not been widely explored although the ECM plays a very important role in bone development. In our research we focused on examining the bone matrix of very young animals due to the intense growth process during the first month of life. Structure images and fluorescence spectra of the bone surface were carried out using confocal fluorescence microscope Eclipse Ti-S inverted CLSM (NIKON, Japan) for compact tibia of healthy 7-, 14- and 28-day-old rat newborns after prenatal zidovudine administration in comparison with control. Spectral features of ECM autofluorescence were analyzed statistically by taking into consideration p < 0.05. The CLSM technique allows for simultaneous examination of the structure and autofluorescence from selected areas of the bone surface. Excessive autofluorescence of ECM after prenatal zidovudine administration influences bone growth incommensurably to the newborns’ age. Therefore the possibility of an additional non-enzymatic mechanism of collagen cross-linking in the first two weeks of life of newborn rats prenatally treated with zidovudine has been considered. Our results suggest that ECM autofluorescence can be an indicator of bone development in the normal and pathological state.