A study was undertaken to evaluate the retention (R F and R M ) and separation (ΔR F, R α F , α and R S ) of buprofezin (B), flubendiamide (F) and imidacloprid (I) using n-hexane-acetone (6.5 : 3.5 v/v) in the case of NP-HPTLC and methanol-water (8 : 2 v/v) for RP-HPTLC as mobile phase. The study revealed that increasing the acetone content in NP-HPTLC and decreasing the water content in RP-HPTLC resulted in high resolution with increase in R F values for B, F and I. ΔR F > 0.04 and R S > 1.5 were achieved for all pairs of compounds (ΔR F(B-F) = 0.35, ΔR F(F-I) = 0.19, ΔR F(B-I) = 0.54, R S(B-F) = 4.12, R S(F-I) = 7.34, R S(B-I) = 2.02 using NP-HPTLC; ΔR F(F-B) = 0.23, ΔR F(I-F) = 0.26, ΔR F(I-B) = 0.49, R S(F-B) = 2.63, R S(I-F) = 2.97, R S(I-B) = 5.92 using RP-HPTLC). Imidacloprid was adsorbed strongly on NP-HPTLC layer and buprofezin on RP-HPTLC layer, as indicated by their high R M values. The maximum absorption of UV for B, F and I was found to be 252, 242 and 276 nm respectively. Stability analysis indicated that these compounds were stable up to 6 h in methanol and on the plates (NP-HPTLC and RP-HPTLC layers). This protocol is useful for toxicologists to detect a mixture of these insecticides in forensic as well as environmental samples.