The analytical method was developed and validated for the quantification of salbutamol sulfate (SS) and ipratropium bromide (IPB) in accordance with the International Council for Harmonization guidelines in its pure form. The chromatographic partition was completed utilizing a blend of acetonitrile:phosphate buffer (30:70 v/v) with the pH scale adjusted to 3.0 using o-phosphoric acid at a flow rate of 1 ml/minute in Luna C-18(2)(150 × 4.6 mm i.d., 5 μm) column. The wavelength for detection was fixed at 212 nm. The SS and IPB showed a standard linearity curve in the range of 2-12 µg/ml, with retention time at 2.4 and 3.8 minutes, respectively. The developed method was reported to be specific, linear (r 2 ≥ 0.999), precise at intraday and interday levels (% relative standard deviation < 2.0%), accurate (% recovery: 96.02%-103.62%), and robust. The limit of detection and limit of quantification for SS was found to be 0.42 and 1.26 µg/ml, while that of IPB was 0.44 and 1.34 µg/ml, respectively. Additionally, the developed method was effectively applied in quantifying SS and IPB from its pure, commercial, and in-house prepared transdermal system to understanding the in-vitro drug release pattern from patches.