Specific fragmentation patterns are fingerprints that allow for unambiguous identification of molecules, for instance in analytical methods. To reveal and possibly control such specific fragmentation, it is essential to understand the physical processes involved during the activation step. We have performed “on-the-fly” (without trapping device) femtosecond (fs) laser activation/mass spectrometry experiments on gas phase protonated reserpine, a model molecular ion for analytical purpose, at different wavelengths and laser pulse intensities. In contrast to collision induced dissociation (CID) or 267 nm fs-laser activation, evidence of non-statistical fragmentation is observed when using 800 nm fs-laser activation. The associated mechanisms are discussed in terms of fragmentation induced by Coulomb repulsion after ultrafast ionization of the protonated molecule. Our results illustrate that the present “on-the-fly” experimental scheme can help in the understanding of the physics behind fs-laser activation.