RNA interference (RNAi) is a useful research tool for the specific deletion, or knockdown, of target genes that can be exploited both in cultured plant cells and in whole plants. In RNAi, hairpin RNA (hpRNA)-transduced lines are used to identify loss-of-function mutations in multi-copy genes with redundant functions in polyploid plant species. Plants transformed with hpRNA exhibit a range of phenotypes resulting from complete knockdown to weak suppression or tissue- and stage-specific knockdown. Functional genomics using proteomic analysis with two-dimensional gel electrophoresis (2-DE) and mass spectrometry provides valuable information about altered levels of expression of specific genes in biological samples. Here, we describe the proteomic analysis of Oryza sativa (Os) thioredoxin m (Ostrxm) knockdown using 2-DE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).