The reported in-house validated method enables quantification of PBT residues from horse meat matrix. With the applied sample preparation method, effective deconjugation and prevention of early degradation of target compound with high recovery and good repeatability was achieved. The sample pretreatment procedure requires two clean-up steps, which ensures clean meat extracts with no matrix effect and enables high sensitive quantification. In-house validation of the method was carried out according to the current European legislation recommendations. Taking into account the current rapidly growing interests in phenylbutazone measurement we have proven that this method is fit for purpose and can be applied for routine testing analysis.