We describe a new thin-layer chromatography (TLC) method to evaluate the radiochemical purity of 99Tc(m)-tetrofosmin without the drawbacks of toxicity, solvent ratios and time requirement associated with the standard TLC method. The new method uses miniaturized instant TLC plates impregnated with silica gel (ITLCTM/SG, 2.5 x 10 cm) for the stationary phase and 2-butanone for the mobile phase. The standard TLC method was performed with ITLCTM/SG plates (5 x 20 cm) and dichloromethane/acetone (65:35, v/v). Thirty five preparations were analysed by both methods with a storage phosphor imaging system to determine the percentages of hydrolyzed-reduced 99Tc(m) compound (99Tc(m)O2), 99Tc(m)-tetrofosmin and free 99Tc(m)-pertechnetate (99Tc(m)O4(-)). Using the miniaturized TLC method, 99Tc(m)-tetrofosmin had a mean Rf value of 0.55 (standard deviation, 0.05), while 99Tc(m)O4(-) migrated with the solvent front (Rf=1) and 99Tc(m)O2 remained at the origin of the strips (Rf=0). No significant difference was found between miniaturized and standard TLC methods for the radiochemical purity of 99Tc(m)-tetrofosmin using the Wilcoxon matched-pair signed-rank test (P=0.82). Furthermore, the two methods showed a good correlation as measured by the Spearman rank coefficient (r=0.89) and were in perfect agreement, with a kappa index of +1, for a cut-point between positive and negative set at 90%. In conclusion, the results indicate that the miniaturized TLC method is effective for the routine evaluation of the radiochemical purity of 99Tc(m)-tetrofosmin, without some of the drawbacks of the standard method.