N,N,N′,N′,N“,N”‐hexaalkylnitrilotriacetoamide (HRNTA) derivatives were found to be promising for the separation of trivalent actinides from lanthanides, which is a burning topic in the back end of the nuclear fuel cycle. In the present work, separation of Am3+ from Eu3+ was studied using the N,N,N’,N’,N’’,N’’‐hexa‐2‐ethylhexylnitrilotriacetamide (H2EHNTA) derivative and the role of the diluent medium to control the extractable complex was demonstrated. The effect of alkyl chain length and branching in the alkyl chain of different HRNTA (R=methyl, n‐butyl, n‐hexyl, n‐dodecyl and 2‐ethylhexyl) derivatives on their complexation with Eu3+ were investigated using steady‐state and time‐resolved luminescence studies. Different species formed at different ligand to metal ratios were identified from the luminescence titration experiments and their lifetime values measured. The experimental results were corroborated with the help of density functional theoretical (DFT) calculations on Am3+ and Eu3+ complexes of HRNTA derivatives with varying alkyl chain length and branching.