The zebrafish owns remarkable regenerative capacities allowing regeneration of several tissues, including the heart, liver and brain. To identify protein dynamics during fin regeneration we used a pulsed SILAC approach that enabled us to detect the incorporation of (13) C6 -lysine (Lys6) into newly synthesized proteins. Samples were taken at four different time points from non-injured and regrowing fins and incorporation rates were monitored using a combination of single-shot 4 h gradients and high-resolution tandem mass spectrometry. We identified more than 5000 labeled proteins during the first three weeks of fin regeneration and were able to monitor proteins that are responsible for initializing and restoring the shape of these appendages. The comparison of lys-6 incorporation rates between non-injured and regrowing fins enabled us to identify proteins that are directly involved in regeneration. For example, we observed increased incorporation rates of two actinodin family members at the actinotchria, which is a hairlike fibre structure at the tip of regrowing fins. Moreover, we used quantitative real-time RNA measurements of several candidate genes, including osteoglycin, si:ch211-288h17.3, and prostaglandin reductase 1 to correlate the mRNA expression to lys-6 incorporation data. This novel pulsed SILAC methodology in fish can be used as a versatile tool to monitor newly synthesized proteins and will help to characterize protein dynamics during regenerative processes in zebrafish beyond fin regeneration. This article is protected by copyright. All rights reserved.