Background: The 20-metre multistage shuttle run test is a useful method for the regular monitoring of aerobic fitness. However, the validity of the test should be established in the particular population prior to application. The aim of the study was to validate the applicability of the 20-metre multistage shuttle run test in non-athlete, girls from Kolkata, India. Methods: Thirty-six untrained girls from different schools in Kolkata (age range 14–16 years) were recruited for the study. Direct estimation of cardiorespiratory endurance (VO2max) comprised treadmill exercise followed by expired gas analysis using Scholander micro-gas analyser. VO2max was indirectly predicted by the 20-metre multistage shuttle run test. Results: The difference between the mean (SD) VO2max values of the direct measurement, 32.91 (2.66) ml.kg-1.min-1, and the 20-metre multistage shuttle run test, 33.79 (2.56) ml.kg-1.min-1, was statistically significant (P < 0.01). However, limits of agreement analysis suggested that the 20-metre multistage shuttle run test can be applied for use with the studied population. Intra-class correlation coefficients also suggested good reliability of the 20-metre multistage shuttle run data. Conclusion: The results suggest that the use of the 20-metre multistage shuttle run test for the prediction of VO2max is justified in the studied population. For better prediction of VO2max, a new equation has been developed based on the present data for untrained girls from Kolkata.