Employing affordable and uncomplicated sample preparation techniques to recommend the most efficient antibacterial therapy could help reduce antibiotic-resistant bacteria. This study evaluated the suitability of immunoassays and microbiological assays as alternatives for liquid chromatography/mass spectrometry (LC/MS) in determining plasma tylosin concentrations after intramuscular administration at a dose of 20 mg/kg to both healthy and diseased pigs in clinical veterinary practice. The diseased pigs were confirmed using the target genesActinobacillus pleuropneumoniae(apxIVA) andPasteurella multocida(kmt1). The methods showed good linearity, precision, and accuracy. In both healthy and diseased pigs, a significant correlation was observed between LC/MS and the microbiological assay (Pearson correlation coefficient: 0.930,p< 0.001 vs. Pearson correlation coefficient: 0.950,p< 0.001) and between LC/MS and the enzyme-linked immunosorbent assay (ELISA) (Pearson correlation coefficient: 0.933;p< 0.001 vs. Pearson correlation coefficient: 0.976,p< 0.001). A strong correlation was observed between the microbiological assay and the ELISA in both healthy and diseased pigs (Pearson correlation coefficient: 0.911;p< 0.001 vs. Pearson correlation coefficient: 0.908,p< 0.001). A Bland-Altman analysis revealed good agreement between the methods, i.e., 95% of the differences were within the limits of agreement. Therefore, the microbiological assay and the ELISA, which demonstrated sufficient precision and accuracy, can be viable alternatives to LC/MS when it is unavailable.