The ability of zebrafish to fully regenerate its caudal fin has been explored to better understand the
mechanisms underlying de novo bone formation and to develop screening methods towards the
discovery of compounds with therapeutic potential. Quantifying caudal fin regeneration largely
depends on successfully measuring new tissue formation through methods that require optimization
and standardization. Here, we present an improved methodology to characterize and analyse overall
caudal fin and bone regeneration in adult zebrafish. First, regenerated and mineralized areas are
evaluated through broad, rapid and specific chronological and morphometric analysis in alizarin
red stained fins. Then, following a more refined strategy, the intensity of the staining within a 2D
longitudinal plane is determined through pixel intensity analysis, as an indicator of density or thickness/
volume. The applicability of this methodology on live specimens, to reduce animal experimentation
and provide a tool for in vivo tracking of the regenerative process, was successfully demonstrated.
Finally, the methodology was validated on retinoic acid- and warfarin-treated specimens, and further
confirmed by micro-computed tomography. Because it is easily implementable, accurate and does not
require sophisticated equipment, the present methodology will certainly provide valuable technical
standardization for research in tissue engineering, regenerative medicine and skeletal biology.