The peptide, octreotide, is used to treat acromegaly and neuroendocrine tumours. It is administered by either a long-acting injection or by an oral capsule. Although the oral route is preferrable, food interference necessitates dosing octreotide on an empty stomach and bioavailability is only ∼0.7%. Buccal film administration avoids the fasting constraints of oral octreotide and offers the potential for improved systemic bioavailability. This study presents a Quality by Design (QbD)- informed strategy to create a mucoadhesive pullulan-based buccal films co-entrapping octreotide with a bile salt permeation enhancer, sodium glycodeoxycholate (GDC). Pullulan was selected from a panel of candidate polymers based on film-forming performance and compatibility with octreotide and GDC, enabling reproducible formulation at relatively high entrapment levels, despite the tendency of them to associate. Atomic force microscopy of films revealed concentration-dependent nanoscale roughness and microdomain formation at high GDC and octreotide levels. However, these structural features did not compromise key critical quality attributes (CQAs) including mechanical robustness, rapid hydration/disintegration, and acceptable content uniformity. Critical performance attributes (CPAs) using isolated porcine buccal mucosae demonstrated that films containing 10% w/w GDC (relative to pullulan) enhanced tissue uptake and octreotide permeation via reversible non-destructive epithelial modulation. Stability testing confirmed >90% chemical recovery and conformational integrity of octreotide in films. UHPLC–HR-MS/MS confirmed identical fragmentation patterns for film-released and native octreotide, confirming the peptide’s intact cyclic backbone and the absence of chemical modification after formulation and after >60 days storage at ambient environment. This is the first report to integrate nanoscale structural analysis with systematic QbD- informed evaluation of formulation quality and performance, establishing pullulan–GDC buccal films as a robust, adaptable platform for future multilayer oromucosal peptide film development.