Pseudaminic acids, a bacterial specific class of nonulosonic acids observed across diverse bacterial phyla, are a common component of capsular polysaccharides, lipopolysaccharides and glycans used for protein glycosylation. The incorporation of this carbohydrate into multiple bacterial glycoconjugates makes the development of Pseudaminic acid-specific affinity tools an attractive approach for both improving our understanding of bacterial glycoproteomes and serving as a potential passive immunity therapy to combat infections. Using synthetically generated glycopeptides as antigens, we have generated and characterized monoclonal antibodies specific for Pseudaminic acid. These antibodies react to both Pseudaminic acid directly attached to proteins and terminally exposed Pseudaminic acids within glycans. Using this tool, we demonstrate that the O-linked glycosylation systems of Campylobacter jejuni and Helicobacter pylori extend beyond the previously reported flagellin substrates, confirming multiple novel O-linked glycoproteins. Extending the application of this antibody to Acinetobacter baumannii glycosylation, we demonstrate that Pseudaminic acid incorporated into glycan chains is detectable in a stereochemistry-specific manner, providing a sensitive means to track alterations in capsule types, visualize infections through immunofluorescence imaging, and expand the known glycoproteome of A. baumannii. Importantly, while Pseudaminic acid-specific antibodies allow for the enrichment of glycoconjugates across multiple contexts, they also highlight the need for improved approaches to track isobaric changes in glycan structures. By leveraging energy-resolved mass spectrometry approaches, we show that isobaric differences in glycan compositions can be distinguished by the unique glycan fragmentation patterns observed in an energy dependent manner. Thus, by combining approaches such as carbohydrate-specific affinity enrichment with energy-resolved MS, this provides new avenues to explore isobaric Pseudaminic acid-containing glycosylation events observed across bacterial pathogens.