The microbiological bioassay, the adenylation method, and the radiometric, enzyme, and fluorescent immunoassay methods for assaying serum gentamicin were compared. The precision, reproducibility, and specificity of each method was assessed and proved satisfactory, with the exception of the radioimmunoassay, which gave artificially high results. Good correlations between the other four methods was obtained in a comparison involving 103 patient sera. The other aspects of performance were also compared, namely, simplicity, speed, cost, ease of automation, and application to large and small workloads. The enzyme immunoassay performed best in this comparison, being accurate, specific, rapid, and very simple to perform. However, other laboratories might find that workload, staffing, and available equipment make other methods more attractive.