Structural elucidation and distinction of isomeric neurotoxic agents remain a challenge. Tandem mass spectrometry can be used for this purpose in particular if a "diagnostic" product ion is observed. Different vibrational activation methods were investigated to enhance formation of diagnostic ions through consecutive processes from O,O-dialkyl alkylphosphonates. Resonant and non-resonant collisional activation and infrared multiphoton dissociation (IRMPD) were used with different mass spectrometers: a hybrid quadrupole Fourier transform ion cyclotron resonance (Qh-FTICR) and a hybrid linear ion trap-Orbitrap (LTQ/Orbitrap). Double resonance (DR) experiments, in ion cyclotron resonance (ICR) cell, were used for unambiguous determination of direct intermediate yielding diagnostic ions. From protonated n-propyl and isopropyl O-O-dialkyl-phosphonates, a diagnostic m/z 83 ion characterizes the isopropyl isomer. This ion is produced through consecutive dissociation processes. Conditions to favor its formation and observation using different activation methods were investigated. It was shown that with the LTQ, consecutive experimental steps of isolation/activation with modified trapping conditions limiting the low mass cut off (LMCO) effect were required, whereas with FT-ICR by CID and IRMPD the diagnostic ion detection was provided only by one activation step. Among the different investigated activation methods it was shown that by using low-pressure conditions or using non-resonant methods, efficient and fast differentiation of isomeric neurotoxic agents was obtained. This work constitutes a unique comparison of different activation modes for distinction of isomers showing the instrumental dependence characteristic of the consecutive processes. New insights in the dissociation pathways were obtained based on double-resonance IRMPD experiments using a FT-ICR instrument with limitation at low mass values.