Although it is well known that there is a significant contribution of precipitation strengthening to the strength of structural steels, it is difficult to experimentally separate the precipitation contribution from the overall strength due to the complex nature of steel microstructures. In this study, the contribution of precipitation strengthening to the matrix strength of ferrite in steel was experimentally investigated through nanoindentation tests. Nanoindentation hardness values of the ferrite matrix in precipitates-containing steels were greater than those in precipitate-free steels, which may be due to the Orowan strengthening mechanism based on the interaction between precipitates and dislocations. To better understand the precipitation strengthening contribution, interparticle spacing values of precipitates were calculated for each microalloyed steel and used for the analysis of hardness change. It was revealed that the trend of nanoindentation hardness change with variation in interparticle spacing corroborated the expectation based on classic Orowan theory.