This study outlines the development and optimization of an analytical method using Disposable Pipette Extraction (DPX) followed by high-performance liquid chromatography-mass spectrometry (LC-MS) analysis of NAs in medicines. LC-MS analysis utilized reverse phase and positive mode electrospray ion sources. DPX parameters were optimized through univariate and multivariate analyses, including extraction phase, desorption solvent, sample pH, equilibrium time, and extraction/desorption cycles. Optimized conditions included a C18 extraction phase, methanol desorption solvent, pH at 7, 30 seconds of equilibrium time, 2 extraction cycles, and 5 desorption cycles. Considering this method, it was possible to achieve a sample preparation step for the analysis of NAs in medicines using a minimal amount of extraction phase, sample, and desorption solvent. Furthermore, the total extraction procedure enables the extraction of NAs in around 4 minutes with NAs recovery of up to 98%. Analytical performance demonstrated precision and accuracy below 15% and a quantification limit of 1 ng mL-1, meeting validation requirements set by regulations worldwide. Thus, the DPX/LC-MS technique offers a faster and more cost-effective method for analyzing NAs in medicines compared to traditional approaches. Besides, this method reduces solvent consumption and residue generation, enhancing environmental sustainability according to green chemistry principles.