This work aimed to evaluate the Mycoplasma Duo kit as a rapid method for detection of ureaplasma in endotracheal aspirate samples from respiratory distressed premature neonates compared to conventional culture media. Also its sensitivity and specificity were determined. This study was carried out on 60 premature neonates (less than 35 gestational weeks) suffering from respiratory distress and mechanically ventilated in neonatal intensive care unit.From all cases paired endotracheal aspirate samples were collected aseptically using an appropriately sized catheter. Endotracheal aspirate samples were transported in ureaplasma transport media to the laboratory and processed immediately. one of each pair of the collected endotracheal aspirate samples were cultured in both Ureaplasma agar and broth cultures and others were cultured in Mycoplasma Duo kit.The number of ureaplasma detected with both ureaplasma agar and broth cultures are 20 cases (33.33%) while those detected by Mycoplasma DUO kit are 22 cases (36.67%).The Sensitivity of Mycoplasma Duo kit compared with both Ureaplasma agar and broth cultures is (100%) and specificity is (95%).There is a highly significant difference (P-value < 0.001) between Mycoplasma Duo kit and Ureaplasma agar and broth cultures as regards the incubation time taken to get positive results by both tests. Mycoplasma Duo kit is simple, rapid ( the results are available in 24-48hrs) and has a sensitivity and specificity comparable to conventional culture methods for detection of ureaplasma spp. The Mycoplasma Duo assay is a useful alternative to culture for detection of neonatal Ureaplasma infection.