Background: Multiplexed bead immunoassays allow simultaneous measurement of adipokines and other hormones in small serum samples, although a validation of this technique with classical methods has not been fully established. The purpose of this pilot study was to compare the characteristics of a multiplexed bead immunoassay obesity panel for insulin and various adipokines with classical methods. Methods: A multiplexed bead immunoassay was performed using serum from 20 obese children at baseline and after reducing their body mass index, and in 25 controls. Insulin, adiponectin, leptin, resistin, tumor necrosis factor-α and interleukin-6 measured by multiplexed bead immunoassay were compared with results obtained from commercial immunoassays. Correlation, sensitivity, recovery, linearity, performance and imprecision were established for each analyte. Results: The correlation between methods was acceptable for adiponectin, leptin, and insulin with coefficients of 0.75–0.89 (p<0.001). Correlation was weak for resistin (0.54, p<0.001) and poor (r<0.30) for tumor necrosis factor-α and interleukin-6. However, Bland-Altman analysis indicated agreement for insulin methods (bias=–0.07), avoiding direct comparison with other analytes (bias>1.25). The imprecision was similar for both methods (<13%). Multiplexed immunoassay had a broader dynamic range than classical methods (4.94 times). The magnitude of the changes in serum concentrations after weight loss was comparable with both methods for adiponectin, leptin, insulin and resistin, resulting in similar statistical significance. Changes in tumor necrosis factor-α and interleukin-6 were detected by classical immunoassays only (p<0.05). Conclusions: This study demonstrates that multiplexed bead immunoassay is more cost effective for measurement of adipokines present in relatively large amounts, diminishing inter-assay variations and reducing the sample volume. Clin Chem Lab Med 2010;48:1439–46.