By using an unambiguous in vivo deuterated-leucine labeling quantitative proteomic approach, at close to the physiologically relevant level, we systematically profiled multiple proteins interacting with 14-3-3epsilon, the isoform with least characterized protein interactions in 14-3-3 family in mammalian cells. Among the 19 proteins interacting with 14-3-3epsilon identified, 6 of them including SKb1Hs, p54nrb, serine/threonine kinase 38, MEP50, 14-3-3theta and cofilin 2 were the previously unknown interacting partners with 14-3-3epsilon. The newly identified interactor cofilin 2 was also validated in co-transfection and co-immunoprecipitation. In contrast, with the same stringent criteria only three known partners were identified by conventional tandem affinity purification (TAP) approach. Therefore the 'in-spectra' quantitative marker of deuterated-leucine assisted to precisely identify those genuine interacting partners with minimum requirement of validation using other molecular approaches.