In order to find a better method than the toluidine blue method, which has already been reported, to predict aminoglycosides ototoxicities in vitro, other methods were investigated. Among the methods examined, acridine orange method, methylene blue method and turbidimetric method, the last was best. As for the stability and sensitivity, however, the toluidine blue method was better than the turbidimetric method so far. Complex formation between polyanions and aminoglycosides reduced the antimicrobial activity of aminoglycosides antibiotics although the concentrations of the polyanions tested were much higher, from 63 to 390 times, than the antibiotics used. This fact, however, must be borne in mind in case of the clinical use of polyanions.