Aminoglycosides (AMGs) are an important class of antibiotics to treat Gram-negative bacterial infections. They can also be used as growth promoters in food-producing animals. AMGs are often analyzed in honey, eggs, milk, tissues and fluids of food-producing animals for control and monitoring purposes. In this study, the effect of chromatographic conditions on the liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS) of common AMGs using a novel zwitterionic hydrophilic interaction chromatographic column are investigated. The effect of a novel hybrid surface technology, which was recently developed to mitigate analyte metal interactions, is also studied. The AMGs included in this study are streptomycin, dihydrostreptomycin, spectinomycin, apramycin, paramomycin, kanamycin, gentamycin C1, gentamycin C2/C2a, gentamycin C1a, neomycin, and neomycin A. The chromatographic conditions under investigation include mobile phase additive and buffer concentration, mobile phase pH, column temperature, and elution gradient. The chromatographic behaviour of the AMGs under various conditions will be discussed.