Octreotide acetate (OA), a potent octapeptide, is used in the treatment of pituitary adenoma. An approach has been made in the present research to formulate an OA-loaded intranasal in situ gel (OA-ISG) to target pituitary adenoma. To achieve the objective of the present work, OA-ISG was fabricated using cold method, and further optimization was done by 32 factorial design. The optimized formulation was evaluated for gelation temperature, mucoadhesive strength, and % drug release (8 h), and the results were found to be 30.01 ± 0.4 °C, 40.12 ± 0.5 g, and 98.54 ± 0.45 %, respectively. Brain availability of OA was determined through gamma scintigraphy, wherein Cmax for technetium (99mTC) labeled intranasal OA-ISG (99mTC-OA-ISG) was found to be 1.041 % RA/g, and the findings for 99mTC-OA-Solution (intranasal) and 99mTC-OA-Solution (intravenous) were 0.395 % and 0.164 % RA/g, respectively. Consequently, a 3–10-fold increase in brain OA concentrations was observed upon intranasal administration (OA-ISG) as compared to others. Additionally, drug targeting index (100.13), targeting efficiency (10013 %), and direct transport percentage (2564.1 %) corroborate brain targeting of OA via intranasal route. Further, the cytotoxic potential of OA-ISG was screened on human pituitary tumor (GH3) cell lines using MTT assay. The IC50 value was found to be 9.5 μg/mL for OA-ISG, whereas it was 20.1 μg/mL for OA-Solution, thereby confirming the superior results of OA-ISG as compared to OA-Solution. Hence, the developed intranasal OA-ISG can be further explored for establishing its potential clinical safety, and as effective platform for targeted drug delivery to the brain in pituitary adenoma. © 2022 Elsevier B.V.