This prospective study was aimed to assess inter- and intra-operator variability during routine quality control (QC) procedure for technetium-99m mercaptoacetyl triglycine ((99m)Tc-MAG3) instant kit formulation. A total of 160 QC analyses with thin layer chromatography (TLC) for 20 separate MAG3 re-constitutions were performed by 2 radiochemists. The percentage of free and hydrolysed (99m)Tc as well as binding efficiency, were calculated according to standard TLC. Each QC analysis was done using silica gel (SG), silica acrylic (SA), Whatman 1 (W1) and Whatman 3 (W3) TLC strips separately at 1h, following labeling MAG3 instant kit with (99m)Tc-pertechnetate. To assess the radiochemical stability of (99m)Tc-MAG3, the same analysis was performed 4h after kit reconstitution. Visual confirmation for QC with scintigraphy was also performed. At both time points, each radio-chemist repeated all the procedure twice for each of the TLC paper types to analyze the intra-operator reliability. Crombach's Test was used for the reliability analysis. High inter-operator correlation ratios (range: 0.821-0.920) per each TLC strip were found where the highest concordance rate was 0.921 for SA. Each TLC strip showed adequate kit reconstitution with acceptable free and hydrolysed (99m)Tc percentages both at 1 and 4 h analyses, along with high binding efficiency values of 94.3 +/- 2.9 and 92.5 +/- 1.9 at 1 and 4 h respectively. Intra-observers reliability showed almost equal high concordance rates (range: 0.888-0.961) for all types of strips. In conclusion, all kinds of ITLC/TLC strips were reliable to assess stability of the radiopharmaceutical at 1 and 4 h while analysis with the SA strip had the highest concordence rate. Inter- and intra-operator QC was also reliable.