Neuropeptides play a major role in maintaining normal brain development in children. Dysfunction of some specific neuropeptides can lead to autism spectrum disorders (ASD) in terms of social interaction and repetitive behavior, but the exact underlying etiological mechanisms are still not clear. In this study, we used an animal model of autism to investigate the role of bee pollen and probiotic in maintaining neuropeptide levels in the brain. We measured the Alpha-melanocyte-stimulating hormone (α-MSH), Beta-endorphin (β-End), neurotensin (NT), and substance P (SP) in brain homogenates of six studied groups of rats. Group I served as control, given only PBS for 30 days; Group II as an autistic model treated with 250 mg PPA/kg BW/day for 3 days after being given PBS for 27 days. Groups III-VI were denoted as intervention groups. G-III was treated with bee pollen (BP) 250 mg/kg body weight/day; G-IV with (LB) (109 CFU/mL) suspended in PBS; G-V with 0.2 g/kg body weight/day Protexin, a mixture of probiotics (MPB); and G-VI was transplanted with stool from normal animals (FT) for 27 days prior to the induction of PPA neurotoxicity on the last 3 days of study (days 28-30). The obtained data were analyzed through the use of principal component analysis (PCA), discriminant analysis (DA), hierarchical clustering, and receiver operating characteristic (ROC) curves as excellent statistical tools in the field of biomarkers. The obtained data revealed that brain levels of the four measured neuropeptides were significantly reduced in PPA-treated animals compared to healthy control animals. Moreover, the findings demonstrate the ameliorative effects of bee pollen as a prebiotic and of the pure or mixed probiotics. This study proves the protective effects of pre and probiotics against the neurotoxic effects of PPA presented as impaired levels of α-MSH, β-End, NT, and SP.