This study presents the development of a sophisticated liquid chromatography-mass spectrometry approach leveraging hydrophilic interaction chromatography (HILIC) for the quantification of kanamycin and spectinomycin in fermentation media. The method was validated per International Council for Harmonisation guidelines, demonstrating robust linearity, precision, and accuracy. To mitigate pronounced matrix effects common to complex fermentation matrices, sample preparation was thoroughly optimized with solid-phase extraction employing MCX sorbent, thereby enhancing recovery rates and minimizing analytical interference. The validated protocol demonstrated high correlation coefficients (R >
0.998), underscoring its robustness and reliability for the accurate quantification of antibiotics in challenging bioprocess environments, providing a valuable analytical tool for bioreactor system monitoring.