The Short Synacthen Test (SST) is the gold standard for diagnosing adrenal insufficiency. It requires invasive administration of Synacthen, venous sampling, and is resource-intensive.To develop a nasally administered SST, with salivary glucocorticoids measurement, to assess the adrenal response.We conducted 5 studies: 4 open-label, sequence-randomized, crossover, pharmacodynamic studies testing 6 doses/formulations and a repeatability study. Additionally, pharmacokinetic analysis was undertaken using our chosen formulation, 500 µg tetracosactide with mucoadhesive chitosan, Nasacthin003, in our pediatric study.Adult and children's clinical research facilities.A total of 36 healthy adult males and 24 healthy children.We administered all 6 nasal formulations using an European regulator endorsed atomization device. The IV comparators were 250 µg or 1 µg SST.We analyzed paired blood and saliva samples for plasma cortisol and salivary cortisol and cortisone.The addition of chitosan to tetracosactide and dose escalation increased peak cortisol response (P = 0.01 and 0.001, respectively). The bioavailability of Nasacthin003 was 14.3%. There was no significant difference in plasma cortisol at 60 minutes between 500 µg Nasacthin003 and 250 µg IV Synacthen (P = 0.17). The repeatability coefficient at 60 minutes was 105 nmol/L for IV Synacthen and salivary cortisol and cortisone was 10.3 and 21.1 nmol/L, respectively. The glucocorticoid response in children was indistinguishable from that of adults.Nasal administration of Nasacthin003 generates equivalent plasma cortisol values to the 250-µg IV SST and, with measurement at 60 minutes of salivary cortisol or cortisone, provides a noninvasive test for adrenal insufficiency.