There is a growing need for easy-to-use, low cost and portable quantitative assays to determine active pharmaceutical ingredients in the pharmaceutical industry. Here, we developed a batch spectrophotometric method and a method employing a paper-based microfluidic device for the estimation of Amoxicillin (AMX) in pure solution and pharmaceutical preparations. The detection depends on the coupling reaction of Amoxicillin with diazotized sulfadimidine (DSDM) in an alkaline medium. The yellow azo dye reaction product was measured at λ_max 425 nm and linearity was observed from 2 to 30 mg L⁻¹ with a detection limit of 0.32 mg L⁻¹ and a quantification limit of 1.2 mg L⁻¹ was found. The reaction was then transferred onto the paper-based microfluidic device and a plateau change in color intensity was found above 10 mg L⁻¹. Thus, the paper-based microfluidic device can be applied for the semi-quantitative determination of Amoxicillin in pure solution and commercial pharmaceutical products for rapid screening.