To further understand the complexation and fragmentation during the extraction process, the formation of 2,9-bis(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-12,4-benzotriazin-3-yl)-1,10-phenanthroline (CyMe₄-BTPhen) complexes with lanthanides (Ln = La, Ce, Nd, Sm, Eu, Yb) and actinides (UO₂²⁺, Th⁴⁺) was observed by electrospray ionization mass spectrometry (ESI-MS) technique and density functional theory (DFT) calculations. Mass spectrometry titrations showed the variation relationship of different complexes in acetonitrile. For lanthanides, the major complexes were 1:2 species ([Ln(L)₂]³⁺ and [Ln(L)₂(NO₃)]²⁺) with a ratio of 1:2, which were observed at the initial addition of Ln³⁺, whereas the species ([Ln(L)(NO₃)₂]⁺) with a ratio of 1:1 was detected when the [Ln]/[L] concentration ratio reached 1.0. For UO₂²⁺ and Th⁴⁺ complexes, 1:1 or 1:2 species ([UO₂L(NO₃)]⁺, Th(L)₂(NO₃)³⁺ and Th(L)₂(NO₃)₂²⁺) were formed. The fragmentation chemistry of both the ligand and the complex cations was characterized in detail by collision-induced dissociation. The fragmentation process of CyMe₄-BTPhen was unfolded sequentially on both sides of the ligand by cleavage of C–C and C–N bonds. DFT calculations provided a detailed analysis of the structures and thermodynamics of those complexes, which indicated that the stable complexes formed in acetonitrile solution were consistent with the ESI-MS results.