<p>The diagnosis of gout was developed through the detection of monosodium urate (MSU) crystals in the synovial fluid of patients with gout using MSU-binding peptides. Amino acid sequences with a high binding affinity to MSU crystals were screened from the Fv-antibody library using MSU crystals as probes. Five CDR3 sequences were successfully screened as MSU-binding sequences from the Fv-antibody library, and these were synthesized into 11-residue peptides. Among these, two MSU-binding peptides (Nos. 1 and 3) showed a significantly higher affinity for preventing the growth of MSU crystals. A detection kit for MSU crystals was developed using MSU-binding peptide No. 3 (biotin-labeled). An assay was performed using samples from patients with gout (<i>n</i> = 23) and healthy controls (<i>n</i> = 29). The sensitivity and specificity of this assay were estimated to be 82.6% and 93.1%, respectively, at a confidence interval of 95%. These results show that the MSU crystal assay based on MSU-binding peptides can be effectively applied for the medical diagnosis of gout. Furthermore, the MSU crystal assay can be carried out using only reagents, without polarized microscopes or trained medical doctors.</p> Graphical Abstract <p></p>