Diperiodatoargentate(III) (DPA)/silver(III) complex, [Ag(HIO6)2] 5-, in sulfuric acid medium has been used to determine hyoscine butylbromide (HBB) by flow injection (FI) coupled with chemiluminescence (CL) detector. A linear standard curve between the CL intensity and concentration range from 0.005 to 20 mg L -1 was obtained. The determination coefficient (R 2 ), limit of detection (3s × blank), relative standard deviation (RSD) for 0.5 mg L -1 HBB and analytical throughput were 0.9992 (n = 8), 5 × 10 -4 mg L -1 , 1.5% (n = 10) and 160 injections h -1 , respectively. The developed method was applied for the determination of HBB in pharmaceutical formulations with recoveries from 92 ± 4 to 108 ± 3%. For comparison, a spectrophotometric method was used and the results obtained by both methods were in good agreement at a 95% confidence level. The effect of key chemical and physical variables (reagent concentration, flow rate, sample volume, PMT voltage) and interfering species (pharmaceutical excipients and inorganic ions) on the determination of HBB was examined. The possible CL mechanism of HBB on silver(III) complex in sulfuric acid medium was also discussed in brief.Keywords Chemiluminescence, flow injection analysis, silver(III) complex, hyoscine butylbromide, pharmaceutical formulations