Digging more missing proteins (MPs) is an important mission of C-HPP. With the MP number is being attenuated year by year, we have realized that the difficulty to explore the remaining MPs is a challenge in technique. Herein, we propose a comprehensive strategy to effectively enrich, separate and identify the proteins with low molecular weight, aiming at discovery of MPs. Basically, the protein extract from human placenta were passed through a C18 SPE column, and the bound proteins eluted were further separated by SDS-PAGE gel or 50kD cutoff filter. The separated proteins were undergone trypsin digestion and the MS/MS signals were searched against datasets with two different digestion modes. The strategy was adopted, resulting in 4 MPs ( ≥2 non-nested unique peptides with ≥9 amino acids) identification containing 8 unique peptides, one of which was only composed of 80 a.a.. Importantly, seven out of eight unique peptides derived from the MPs were verified by parallel reaction monitoring, demonstrating that the placenta tissues indeed possessed the MPs. The study does not only establish a feasible strategy for analysis of the proteins with low molecular weight, but also fill a small gap to the MP list.