Prokaryotes are known to produce and secrete a broad range of biopolymers with a high functional and structural heterogeneity, often with critical duties in the bacterial physiology and ecology. Among these, exopolysaccharides (EPS) play relevant roles in the interaction of bacteria with eukaryotic hosts. EPS can help to colonize the host and assist in bacterial survival, making this interaction more robust by facilitating the formation of structured biofilms. In addition, they are often key molecules in the specific recognition mechanisms involved in both beneficial and pathogenic bacteria-host interactions. A novel EPS known as MLG (Mixed-Linkage β-Glucan) was recently discovered in rhizobia, where it participates in bacterial aggregation and biofilm formation and is required for efficient attachment to the roots of their legume host plants. MLG is the first and, so far, the only reported linear Mixed-Linkage β-glucan in bacteria, containing a perfect alternation of β (1 → 3) and β (1 → 4) bonds. A phylogenetic study of MLG biosynthetic genes suggests that far from being exclusive of rhizobia, different soil and plant-associated bacteria likely produce MLG, adding this novel polymer to the plethora of surface polysaccharides that help bacteria thrive in the changing environment and to establish successful interactions with their hosts.In this work, a quantification method for MLG is proposed. It relays on the hydrolysis of MLG by a specific enzyme (lichenase), and the subsequent quantification of the released disaccharide (laminaribiose) by the phenol-sulfuric acid method. The protocol has been set up and optimized for its use in 96-well plates, which makes it suitable for high-throughput screening (HTS) approaches. This method stands out by its fast processing, technical simplicity, and capability to handle multiple samples and biological replicates at a time.