Aggregates of misfolded α-Synuclein in the brain represent a hallmark of Parkinson’s disease (PD). In patients and animal models, phosphorylated α-Synuclein was detected in the gut, hence, raising the hypothesis that early-stage PD could be diagnosed based on colon tissues. Marker-independent technologies represent an ideal method to monitor disease progression and potentially detect early-stage aggregated α-Synuclein in vivo. Here, formalin-fixed, paraffinembedded colon tissues of a transgenic rat model were analyzed using Raman imaging. Detailed spectral and imagebased analysis was performed indicating the major spectral shifts that alter in PD rat tissues in the amide I region. Peak fitting and multivariate analysis specified an increase of β-sheet proteins in transgenic rat colon compared with wild-type colon. In summary, Raman imaging is capable to detect α-Synuclein aggregates in colon tissues of a PD rat model, indicating that it could be a useful tool to support diagnosis in PD pathology.