The purpose of this study was to develop a sublingual drug delivery spray formulation of scopolamine hydrobromide (L-(-)-hyoscine hydrobromide) and to determine the absolute bioavailability of scopolamine hydrobromide following sublingual delivery and to investigate the effect of a bioadhesive on the pharmacokinetic parameters of this drug in a rabbit model. Rabbits received a single scopolamine free base equivalent sublingual dose of 100 microg/kg and this was compared to intravenous administration of the drug. Blood samples were collected at different time points, and plasma scopolamine concentrations were determined using a new sensitive and specific LC/MS analytical method which utilized electrospray ionization detection. The bioavailability of sublingual scopolamine was determined by comparing plasma concentrations after sublingual spray delivery with equivalent intravenous doses. Following delivery of the sublingual spray dose, the average Cmax was 1024.4+/-177 ng/mL, and the AUC value was found to be 61067.6+/-9605 ng.min/mL. Relative to the intravenous dose (100% bioavailability), the bioavailability was 79.8% after sublingual spray administration. The addition of 2% chitosan, a bio-adhesive material and an absorption enhancer, showed a significant improvement in scopolamine sublingual absorption (p<0.05) was observed. Considering the limitations of delivering scopolamine orally or transdermally to patients who experience motion sickness, the sublingual route of administration using a spray delivery dosage form, is a potential alternative modality for the prevention of nausea and vomiting associated with motion sickness.