The study of multivalent carbohydrate–protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. Bio-Layer Interferometry (BLI) is emerging as a tool to monitor such complex interactions. In this study, various glycoclusters and dendrimers were prepared and evaluated as ligands for lectins produced by pathogenic bacteria Pseudomonas aeruginosa (LecA and Lec B) and Burkholderia ambifaria (BambL). Reliable kinetic and thermodynamic parameters could be measured, and immobilization of either lectin or ligands resulted in high quality data. The methods gave results in full agreement with previous ITC experiments, and presented strong advantages since they require less quantity and purity for the biomolecules.