The in vitro pepsin digestion assay is the international standard for assessing the safety or risk of novel proteins newly produced in transgenic crops. However, this protocol, based on the degradation of protein purified from Escherichia coli, has recently been criticized for problems such as its objective detection limit. Here, we estimated the digestion stability of the phosphinothricin acetyltransferase (PAT) protein in soluble proteins as well as from leaf tissue powder in simulated gastric fluid (SGF). Our line of genetically modified zoysiagrass carried a single copy of the bar gene, which entered a chromosomal region not encoding protein. We designated it as Jeju Green 21 (JG21). From total soluble proteins extracted from JG21 leaves, digestibility of the PAT protein in SGF was examined by enzymatic assays, sodium dodecyl sulfate.polyacrylamide gel electrophoresis, Western gel blots, and an immunodetection strip kit. The degradability of pure PAT protein obtained from E. coli was clearly apparent within at least 30 s. However, PAT degradation in leaf tissue powder was significantly delayed, indicating that some matrices in that powder might have influenced its digestion stability by SGF. Nevertheless, degradation of the powder (real-life) sample was complete within at least 5 min, suggesting that this protein produced in JG21 zoysiagrass can be digested harmlessly in the stomachs of humans or livestock.