Polyclonal and monoclonal antibodies against valnemulin, a new semi-synthetic antibiotic derivative of pleuromutilin administered to treat swine dysentery and pneumonia, were generated. To achieve high enzyme-linked immunosorbent assay sensitivity for valnemulin, several heterologous coating antigens were prepared and evaluated, differing in the length of the spacer arm and the conjugation site between valnemulin and carrier protein. After the optimisation of immuno-reagents dilution, the enzyme-linked immunosorbent assay, based on polyclonal antibody number 2 and one heterologous coating antigen, showed the highest sensitivity with an IC(50) value for valnemulin of 0.96 ng/mL in buffer. For spiked porcine liver, an extraction procedure with a mixture of acetonitrile and 0.01 M hydrochloric acid (40:60 v/v) was proposed and no further sample pre-treatment other than 10 times dilution of the extract was necessary prior to analysis, which gave recovery values ranging from 75.7% to 89.4%. The dynamic assay range and the limit of detection of the assay were 2.4-49.9 and 1.67 µg/kg for porcine liver, respectively. The assay was compared with a confirmation method based on LC-MS/MS by using valnemulin-treated samples, and a satisfactory correlation between both methods was observed.