A simple, inexpensive and efficient agar diffusion bioassay, applying cylinder plate method, for determination of josamycin (JOS) in pure and pharmaceutical dosage forms was studied. Using a strain of Micrococcus luteus ATCC 9341 as the test organism, JOS at concentrations ranging from 0.1 to 0.5 µg/mL could be measured. The calibration graph was linear with good correlation coefficient (r = 0.999), and the method validation showed that it was specific and robust. The interference effects of degradation products and excipients were investigated in order to assess the method selectivity. The proposed method was successfully applied to estimate JOS content in tablets and suspension with good precision (intra-day RSD=0.87%; inter-day RSD=1.25%) and accuracy; the recovery values were between 97.74 and 101.13%. Bioassay was correlated with UV spectrophotometric and high performance liquid chromatographic techniques. The results obtained by these three methods were statistically analyzed by analysis of variance (ANOVA), which indicated that there was no significant difference among these methods. Therefore, we recommend this microbiological assay for drug analysis in routine quality control of josamycin in pharmaceuticals as an alternative to the physicochemical methods reported in the literature.