Providing regular external quality assessment of primary level laboratories and timely feedback is crucial to ensure the reliability of testing capacity of the whole laboratory network. This study was aimed to assess the diagnostic performances of primary level laboratories in Southwest Showa Zone in Ethiopia. An external quality assessment protocol was devised whereby from among all the samples collected on-site at 4 health centers (HCs), each HC sent to a district hospital (DH) on a weekly basis 2 TB slides (1 Ziehl-Neelsen stained and another unstained), 2 malaria slides (1 Giemsa stained and another unstained), and 2 blood samples for HIV testing (1 whole blood and another plasma) for a comparative analysis. Similarly, the DH preserved the same amount and type of specimens to send to each HC for retesting. From October to November 2011, 192 single-blinded specimens were rechecked: 64 TB slides, 64 malaria slides, and 64 blood specimens for HIV testing. The analyses demonstrated an overall agreement of 95.3% (183/192) between the test and the retest, and 98.4% (63/64), 92.2% (59/64,) and 95.3% (61/64) for TB microscopy, malaria microscopy, and HIV rapid testing, respectively. Of the total TB slides tested positive, 20/23 (87%) were quantified similar in both laboratories. The agreement on HIV rapid testing was 100% (32/32) when plasma samples were tested either at HC (16/16) or at DH (16/16), while when whole blood specimens were tested, the agreement was 87.5% (14/16) and 93.8% (15/16) for samples prepared by HCs and DH, respectively. Results of this new approach proved that secondary laboratories could play a vital role in assuring laboratory qualities at primary level HCs, without depending on remotely located national and regional laboratories to provide this support.