Introduction: Amoxicillin and Ampicillin are among the most widely used antibiotics for treating bacterial infections. While traditional drug monitoring methods often face challenges relative to accuracy and analysis speed, optical-based techniques offer a promising alternative. Fourier transform infrared spectroscopy (FTIR), a well-established tool, is particularly suited for this purpose. This study introduces a novel approach to the standard methods of antibiotic detection and monitoring, leveraging the capabilities of vibrational spectroscopy and helping in antimicrobial stewardship. Methods: Principal Component Analysis (PCA) was used in the fingerprint region to detect differences between the studied antibiotics. Additionally, absorbance intensity in the fingerprint region was monitored to assess the degradation of each antibiotic over time. To achieve this, the area under the curve was calculated and subjected to inferential statistical tests for both intragroup (degradation of the same antibiotic) and intergroup (degradation within the same time interval, comparing the two antibiotics) comparisons. All analyses were performed in OriginLab and using Python in the Google Colab and Orange environment. For the calculations of the limit of detection (LoD), the method based on the calibration curve was used. Results: Through the experiments, it was possible to identify the fingerprints of each antibiotic and statistically separate them, despite both belonging to the same class of antibiotics, where the spectral peaks appear in the same region. For degradation, all tests were conducted with a significance level of α = 5%. In this investigation, our results show several quantification characteristics with a detection limit of 186.40 mM for Ampicillin (using peak ratio) and 81.40 mM for Amoxicillin (using peak intensity). Conclusions: This research demonstrates that FTIR spectroscopy is effective for antibiotic detection and has the potential to be further developed into a monitoring protocol.