The effect of laser pulse duration on the ablation of aqueous myoglobin is investigated using laser electrospray mass spectrometry (LEMS). Pulse durations of 55 femtoseconds (fs), 56 piscoseconds (ps), and 10 nanoseconds (ns) were used to ablate aqueous myoglobin from stainless-steel and quartz substrates. The integrated signal intensity of myoglobin increases with decreasing pulse duration for both substrates. Laser-induced thermal effects are assessed by the relative amount of solvent adduction and number of phosphate moieties adducted to myoglobin by each laser pulse duration. The mass spectra for 55 fs vaporization shows myoglobin with appreciable solvent and phosphate adduction and baseline elevation. The mass spectra for 10 ns ablation have minimal adduction and limited baseline elevation. Heat-induced conformation changes in myoglobin were used to measure the amount of thermal energy deposited by each laser pulse duration. Ablation using the 55 fs pulse revealed the highest ratio of unfolded to folded myoglobin in comparison to the 56 ps and 10 ns measurements due to increased droplet lifetime and consequent interaction with the acid in the electrospray solvent. Collisional activation and heated capillary temperature were employed to reduce the droplet lifetime and demonstrate that fs ablation preserves approximately 2 times more myoglobin folded conformation in comparison to ps and ns pulses.