We present the conformational investigations and excited state dynamics of jet-cooled acetanilide derivatives, substituted at ortho positions, such as 2-aminophenol (2-AP), 2-hydroxyformanilide (2-HFA) and 2-acetaminophenol (2-AAP). The number of conformers and their structures of the molecules are assigned on the basis of resonance enhanced multiphoton ionization (REMPI), ultraviolet-ultraviolet hole burning (UV–UV HB), and infrared-dip (IR-dip) spectroscopy. These molecules facilitate to have fast decayed excited state lifetime due to the intramolecular hydrogen bonding interactions between two adjacent atoms. We could not observe these structures with typical nanosecond laser studies. However, we were able to monitor the fast decayed ortho-substituted acetanilide derivatives using population transfer infrared gain spectroscopy. The structures of these conformers are compared with the predictions of ab initio and density functional theory calculations.