Infrared laser ablation sample transfer (LAST) was used to collect samples from solid surfaces for mass spectrometry under native spray conditions. Native mass spectrometry was utilized to probe the charge states and collision-induced unfolding (CIU) characteristics of bovine serum albumin (BSA), bovine hemoglobin (BHb), and jack-bean concanavalin A (ConA) via direct injection electrospray, after liquid extraction surface sampling, and after LAST. Each protein was deposited from solution on solid surfaces and laser-ablated for off-line analysis or sampled for online analysis. It was found that the protein ion gasphase charge-state distributions were comparable for direct infusion, liquid extraction, and laser ablation experiments. Moreover, calculated average collision cross section (CCS) values from direct injection, liquid extraction, and laser ablation experiments were consistent with previously reported literature values. Additionally, an equivalent number of mobility features and conformational turnovers were identified from unfolding pathways from all three methods for all charge states of each protein analyzed in this work. The presented work suggests that laser ablation yields intact proteins (BSA, BHb, and ConA), is compatible with native mass spectrometry, and could be suitable for spatially resolved interrogation of unfolding pathways of proteins.