BACKGROUND: In the year 2014, Nnaemeka and Iyioku in Nigeria developed the methylene blue screening test and advocated for further study regarding the evaluation and usage of the test in a low-resource setting. In this study, we aimed to establish a clear cut-off absorbance for a good predictive value, evaluate the sensitivity and specificity of the test, compare the results to available screening methods and determine the applicability of the test in screening for bacteriuria that does not adhere to Kass criteria and hence thoroughly evaluate the test for external validation before use in low-resource settings. MATERIALS AND METHODS: In this study, a total of 217 urine samples were run through the modified methylene blue test and culture, i.e., the gold standard for diagnosis of significant bacteriuria; using the results, a cut-off was derived using receiver operating characteristic curve analysis. Statistical analysis was then performed using the cut-off. The minimum colony count was determined for the given cut-off to predict whether the test can be used in different settings of bacteriuria. The above results were then compared to available screening tests based on a thorough literature search on PubMed. RESULTS: Using 0.36 as a cut-off absorbance value, the calculated sensitivity and specificity of the test were 83.5% and 92.9% with a positive predictive value of 90% and a negative predictive value of 87.40% according to the current prevalence of urinary tract infection in South India. The P value of the above result determined using the Chi-square test was <0.05 and hence the results were deemed to be statistically significant. CONCLUSION: The test could precisely diagnose bacteriuria with a colony count of >10⁵ colony-forming units/mL and had comparable sensitivity and specificity to a combination of leucocyte esterase and nitrite tests which are popularly used as screening methods.