Due to the highly anisotropic nature of π -conjugated molecules, the molecular structure of organic semiconductors can significantly affect the device performance of organic optoelectronics. Here, the molecular structure dependence on charge injection and doping efficiencies is investigated by characterizing the typical hole transport material of N,N′-bis(naphthalen-1-yl)-N,N′-bis(phenyl)-benzidine (NPB) and its derivatives N,N′-bis(naphthalen-1-yl)-N,N′-bis(phenyl)-9,9-dimethyl-fluorene (DMFL-NPB) and N,N′-bis(naphthalen-1-yl)-N,N′-bis(phenyl)-9,9-diphenyl-fluorene (DPFL-NPB)]. Using photoelectron spectroscopy data and density functional theory calculation, it is identified that the side chain substitution in NPB and its derivatives plays a crucial role in the intrinsic injection and transport properties, and doping efficiency. The inner twist of the two main benzene rings in NPB is changed from out-of-plane to in-plane due to the alkyl or phenyl side chains of DMFL-NPB or DPFL-NPB, which reduces the ionization energies and thus decreases the hole injection barriers at the indium tin oxide/organic interface. The doping efficiency in 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4-TCNQ) doped systems is also highly dependent on the degree of intermolecular orbital energy hybridization with respect to the side chain substitution. These findings show that the rational design of molecular structures with suitable side chains is crucial for achieving high-performance organic devices.