Aztreonam, a synthetic beta-lactam antibiotic is widely used for the treatment of infections in lungs, meninges, bladder, etc. Its potential use in the treatment of pneumonia and cystic fibrosis has been established. Recently FDA has approved Cayston® for inhalation therapy of Aztreonam in lungs infection. Many studies have been reported on the quantitative determination of Aztreonam by UV spectrophotometric and HPLC method. The present study focuses on the quantitative spectrophotometric determination of Aztreonam in two simulated lungs fluid namely artificial lysosomal fluid (ALF) and gamble solution. The λ max was found at a wavelength of 293 nm for both the solutions. Further, the method developed was validated for its linearity, precision by interday and within day study, accuracy, specificity, robustness and determination of limit of quantification and limit of detection in both the solutions. The linearity demonstrated a correlation coefficient of 0.9995 and 0.9999 in ALF and gamble solution respectively. The LOD was found to be 0.38 µg/ml and 0.18 µg/ml for ALF and gamble solution respectively. The LOQ was found to be 1.15 µg/ml and 0.53 µg/ml for ALF and Gamble solution respectively. The proposed method was found to be simple, rapid, accurate, precise, and specific for the determination of Aztreonam in simulated lungs fluid.