Kidney failure is a degenerative disease that has a prevalence of nearly 12 to 15% of the total population in the world. Renal scans are one of many diagnostic means which refers to several examinations using radiopharmaceuticals that evaluate the function and anatomy of the kidneys. Technetium-99m-diethylenetriaminepetaacetic acid ([99Tc]Tc-DTPA) is a commonly used radiopharmaceutical for kidney scans. Since the half-life of technetium-99m radioisotope, one component of this radiopharmaceutical, is only about 6.0 hours, so an efficient and effective quality control techniques is a necessity. Up to now, two-systems thin layer chromatography (TLC) is a common method used for radiochemical purity (RCP) test of [99mTc]Tc-DTPA. Recently one-system TLC has been successfully developed for this purpose. Therefore prior to its application, it is necessary to validate as well to compare this method with an established method (WHO Pharmacopoeia). These methods were two-system used Whatman-1 paper as static phase and methyl ethyl ketone (two-system-A) and 0.9% sodium chloride (two-system-B) as mobile phases and one-system used Whatman-1 paper as static phase and acetone: 0.9% sodium chloride (11:9) as a mobile phase. RCP of [99mTc]Tc-DTPA was then tested using these methods. The retrieved data were processed and validated with some variables like accuracy, precision, and compared using t-test to see whether the above-mentioned TLC systems show a significant difference or not. The analysis results of RCP test of [99mTc]Tc-DTPA using two-system method was 99.37± 0.48% while using one-system method was 99.20 ± 0.41%. The percentage accuracy of the data between two methods was 99.83%. The t-stat value for both methods was 0.84 so it can be concluded that the results of measurement using one-system method are not significantly different from two-system method.