Recombinant bovine somatotropin (rbST) is used to increase milk yield in cows, but it has been forbidden in some countries and in the EU. However, rbST misuse represents a concern in both bovine and buffalo dairy production. A number of studies on rbST treatment have been performed on bovines, but there are few data on buffaloes. In this study, we treated eight lactating buffaloes with biweekly injections of a slow-release formulation of rbST, for five cycles of administration, and analysed total ST and insulin-like growth factor 1 (IGF-1) variations in serum and IGF-1 in milk. The aim was to assess their power as potential indicators of rbST-treatment. Blood was collected on days 2, 5, 9 and 14 of each cycle, and milk on days 2, 9 and 14 of cycles 2 and 5. Results showed an extraordinary increase in ST levels on day 2 in treated animals, followed by a rapid decrease over the following days, while a significant increase in IGF-1 was observed both in serum and in milk throughout most of each cycle. These results suggest that serum ST levels are a good indicator of treatment. However, the rapid decrease after the peak limits the useful period of sample collection.