This protocol describes an immunoassay, originally developed to quantitate ubiquitin in mouse tissues (Oh et al., Anal. Biochem. 443, 153\u2013155), which we adapted to Drosophila melanogaster. The method is suitable for the simultaneous determination of total, free and conjugated ubiquitin forms from whole protein extracts by densitometric analysis of Western blots. In this assay, endogenous deubiquitylating enzymes, DUBs, present in the lysates process all conjugated ubiquitins to monoubiquitins, therefore the total ubiquitin content of cell lysates can be determined in the form of monoubiquitins. The free monoubiquitin fraction in turn is determined from similar lysates, but supplemented with a potent DUB inhibitor, NEM. Appropriate samples of these lysates are immunoblotted together with ubiquitin standards that allow the quantification of the different ubiquitin forms by densitometric analysis of the 8,5 kDa monoubiquitin band.