Assessment of in vivo potency has been a bottleneck for the identification and development of new therapeutics targeting insulin resistance for the treatment of type 2 diabetes. Here we aimed to develop and validate a simplified technique for screening of altered insulin action in conscious and non-constrained mice. The technique was based on measurements of glucose tolerance and glucose tracers under the condition of maximised insulin levels to nullify the impact of possible differences in plasma insulin levels. We first performed ip injection with various doses of insulin (0–9U/kg body weight) supplemented with a fixed dose of glucose in C57BL/6 mice fed with a chow (CH) or high-fat (HF) diet for three weeks. The results showed that this technique was sensitive in detecting dose-dependent stimulatory effects of insulin on glucose tolerance (as indicated by incremental area under the curve [iAUC]). HF-fed mice displayed significant reductions (40-60%, p<0.05) in glucose tolerance when insulin ≥ 1.5U/kg and the stimulating effect of ip insulin on glucose tolerance reached a plateau at 5U/kg in both CH and HF mice. We then examined whether administering 3H-2DG and 14C-glucose tracers would allow assessment of differences in insulin responsiveness between CH and HF mice at 5U/kg of insulin. The results showed that the plasma tracer profiles (AUC) of the glucose tracers displayed similar reductions as that of cold blood glucose (r=0.91 with 3H-2DG and 0.94 with 14C-glucose, both p<0.05). Further measurement of 3H-2DG in skeletal muscle demonstrated insulin-stimulated clearance of this tracer was blunted in HF versus CH mice (57±3 vs 45±3 μl/g/min, p<0.05). Collectively, our data indicate this novel technique is an easily applicable tool for the assessment of insulin action in conscious and non-constrained mice. The technique can potentially enhance the screening of anti-diabetic drugs at the whole body level.