The aim of the present project was to identify putative substrates of Ariadne-1 (Ari-1), an essential E3 ubiquitin-ligase, in Drosophila heads. For that purpose we combined an in vivo ubiquitin biotinylation strategy termed bioUb with label free mass spectrometry analysis. In brief, BioUb strategy relies on the overexpression of a tagged ubiquitin that bears a 16 amino-acid long biotinylatable peptide, which can be biotinylated by the E.coli biotin holoenzyme synthetase enzyme (BirA) in neurons in vivo. Hence, ubiquitinated and therefore, biotinylated proteins can be enriched using avidin prior to MS analysis. We performed the same procedure in triplicate using flies expressing BioUb alone (control) and together with Ari-1. From the 1452 proteins quantified, 16 fulfilled the criteria established to be considered as putative Ari-1 substrates. Additionally, our analyses resulted in the detection of 150 peptides containing the characteristic GlyGly remnant of ubiquitin. Of those, 86 are novel ubiquitination sites not previously described in Drosophila.