Interfacial cross-linking (ICL) has been considered a feasible technique to produce polysaccharide-based microparticles (PbMs), even though only a few studies have been concerned with their biocompatibility. In this work, PbMs were prepared by the ICL method and characterized in regard to their in vitro biocompatibility, chemical linkages, and physical and thermal properties. First, the cell viability assay revealed that PbMs toxicity was concentration-dependent. Then, it was observed that the toxicity may be related to the way in which the binding occurred, and not exclusively to the stoichiometry between the polymer and the cross-linking agent. Moreover, the PbMs biosafety was predicted by the use of physicochemical procedures, which were able to identify unbound cross-linking agent residues and also to reveal the improvement of their thermal stability. Accordingly, this work suggests a step-by-step physicochemical procedure able to predict potential toxicity from micro-structured devices produced by polysaccharides. Likewise, the use of PbMs as a drug carrier should be cautiously considered.