Technical Abstract: This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-fluoroquinolone and anti-sulfonamide broad-specificity monoclonal antibodies (Mabs) to Sepharose 4B for simultaneously isolating 13 fluoroquinolones (FQs) and 6 sulfonamides (SAs) from swine and chicken muscle tissues, followed by antibiotic determination using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A new broad-specificity Mab (B1A4E8) toward sulfonamides was produced using sulfamethoxazole (SMX) as hapten that demonstrated cross-reactivities to 6 SAs in the range of 31–112%. The Mab was used to investigate a correlation between antibody binding and conformational and electronic properties of 6 SA analogs. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of both SAs and FQs. The dual-broad-specificity IAC selectively retained a total of 13 FQs and 6 SAs and allowed for efficient removal of interfering sample matrix compounds. Recoveries of all 19 antibiotics from animal muscle ranged from 72.6–107.6%, with RSDs below 11.3% and 15.4% for intra-day and inter-day experiments, respectively. The limit of quantification (LOQ) ranged from 0.5–3.0 ng/g. The strategy used here for a mixed-bed IAC may be used to study other compounds and more than two classes of analytes simultaneously.