In this work, pendant drop techniques and molecular dynamics (MD) simulations were employed to investigate the effect of asphaltene concentrations on the interfacial tension (IFT) of the oil/water interface. Here, oil and asphaltene were represented by, respectively, common organic solvents and Violanthrone-79, and two types of concentration, i.e., bulk concentration and surface concentration, were examined. Correlations between the IFTs from experiments and MD simulations revealed that surface concentration, rather than the commonly used bulk concentration, determines the reduction of oil/water IFTs. Through analyzing the hydrogen bonding, the underlying mechanism for the IFT reduction was proposed. Our discussions here not only enable the direct comparison between experiments and MD simulations on the IFTs but also help with future interfacial studies using combined experimental and simulation approaches. The methodologies used in this work can be extended to many other oil/water interfaces in the presence of interfacially active compounds.