This paper describes the in vitro DNA end-joining test, a biochemical-molecular method by which, using cell extract or protein fraction derived from tissue with targeted inactivation of genes involved in DNA-damage repair pathways, we can detect mechanisms of DNA double-strand break repair (DSB). An In vitro determination of DNA break repair mechanisms by end-joining is based on synthetic duplex oligonucleotide sequences of various DNA-end configurations with the possibility to be joined. After incubation of linear plasmid DNA as end-joining substrates in a plant cell-free extracts as a source of DNA repair enzymes, and subsequent analysis of structure of the reconstructed DNA ends by agarose gel electrophoresis, or determination of the sequence of nucleotides on junction sites by PCR and DNA sequencing, it is possible to assess the fidelity of end-joining reaction. From this it can be concluded which pathways participate in these end joining events, whether DSB is repaired by a precise or "classical" nonhomologous end-joining (NHEJ) repair pathway, alternative NHEJ (alt-EJ) or microhomologymediated NHEJ (MMEJ). It is also possible to identify core proteins involved in specific DNA repair process.