ABSTRACT Since 2012, Chromosome-Centric Human Proteome Project (C-HPP) launched the journey of missing proteins (MPs) investigation for completing human proteome project (HPP). We found the majority of MPs were distributed in low molecular weight (LMW) ranges, especially in 0-40 kDa. The identification of LMW proteins is challenging owing to their low length, low abundancy, and hydrophobicity. Even worse, many sequences from Trypsin digestion are unlikely to yield detectable peptides or composed less number of observable peptides in MS detection. Therefore, we focused on small MPs by combining LMW protein enrichment and mirror-protease strategy on human testis samples. The in-depth testis LMW protein profiling documented 4,063 proteins from which 2,565 proteins were belong to LMW proteins and 1,130 proteins had mirror peptides. This provided mass spectral evidence for small MPs further verification, and two MPs were verified in seven identified MPs. For Q8N688, confident peptides were verified from Trypsin and LysargiNase digestions, and continuous b/y-ion peptide was obtained by mirror-spectrum matching. Two verified peptides of Q86WR6 were from gel-separation and gel-elution dataset, respectively. And the other 5 MPs cannot be distinguished sufficiently as PE1, but need more verification information.