DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry–based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologous-recombination–based protein tagging on a multi-gene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.