Background: Tuberculosis (TB) is a major public health problem in Ethiopia. Laboratory plays an important role inrnthe diagnosis of TB and its treatment monitoring. Quality assured molecular drug susceptibility assay is an essentialrnfor rapid detection of MDR-TB cases and early treatment initiation Proficiency testing is one of the EQA methods inrnevaluating the quality service delivery however, it was not widely applied for molecular assays in Ethiopia mainlyrndue to the inadequate capacity of preparing PT DNA panelsâ€™ in-country.rnObjective: The aim of this study was to assess Application of Homemade and validated panel DNA Materials forrnProficiency Testing of the WHO endorsed Molecular Assays (GenoTypeÂ® MTBDRplus and Xpert MTB/RIFrnassays) in TB Culture and DST laboratories, and in selected GeneXpert sites of Ethiopia.rnMethods: A cross sectional study was conducted in five regional reference laboratories and two specialized hospitalrnTB culture and DST laboratories which have molecular GenoTypeÂ® MTBDRplus assay of Ethiopia, and in eightrnselected health facilities with Xpert MTB/RIF testing sites in Addis Ababa during the period between July 2015 andrnJanuary 2016. PT DNA materials were validated at EPHI, NTRL and distributed to MTBDRplus and Xpert MTB/RIFrntesting facilities. All data from participating laboratories were captured and analyzed using SPSS version 20. A totalrnof 194 (130 for MTBDRplus and 64 Xpert MTB/RIF) DNA panels were distributed to regional reference and hospitalrnTB laboratories. Test results agreement between the participant laboratories and reference were estimated usingrnKappa statistics. The sensitivity, specificity, predictive value susceptible, predictive value resistance, accuracy andrnreproducibility of participant laboratories were calculated against the reference lab.rnResults: 130 panel samples with a combination of 65 (50%) spiked sputum and 65 (50%) extracted DNA were testedrnin seven Regional Reference Laboratories which have MTBDRplus assay. Almost all, 129 (99.2 %) were correctlyrnreported to the Isoniazid and Rifampicin susceptibility. Sixty nine (53.1%) reported isolates were resistance to bothrnINH and RIF (MDR), 41 (31.5%) susceptible to both INH and RIF, and 19 (14.6%) INH susceptible but RIFrnresistance strains correctly reported with respect to the reference result. There was 100% agreement in detectingrnMDR and resistance to any of the two drugs (INH and RIF) (kappa = 1) but the agreement was 97.8% for therndetection of susceptible strains (Kappa = 0.9). Among 64 spiked sputum strains (32 M. tuberculosis DNA with nornRIF resistance and 32 M. tuberculosis DNA with RIF resistance (probe E Mutant detected ) distributed to GeneXpertrnsites, all of them were correctly reported. Overall agreement on detection of M. tuberculosis and RIF susceptibilityrnwas 100 % (Kappa =1.0).rnConclusion: There was an excellent agreement between the participant laboratories and the reference laboratory forrnthe detection M. tuberculosis and drug susceptibility using MTBDRplus and Xpert MTB/RIF assays. Therefore, werncan apply home validated PT DNA panels as one of the EQA methods for WHO endorsed molecular assaysrnMTBDRplus, whereas in the Xpert MTB/RIF assay a large scale validation is important to use as Panel test since,rnthe sites we used for this study in Addis Ababa, are small compared to the general population in Ethiopian context.