We constructed a yeast‐based reporter assay system to verify the cleavage activity of Arabidopsis thaliana signal peptide peptidase, a transmembrane protease localized in the endoplasmic reticulum. Arabidopsis thaliana signal peptide peptidase cleaved 15 candidate substrates that lacked the positively charged amino acids, His and Lys, in the C‐region of signal peptides. Our assay system may be suitable for identifying innate substrates with crucial roles in plants.