Alpha-chloralose (AC) is used as a rodenticide as well as an anaesthetic agent in laboratory animals. It was previously also used as an avicide. Detection of AC in blood samples or post-mortem in body tissues is key for diagnosis of clinical cases and a requirement for surveillance of secondary toxicosis, including potential cases in wild animals. Reports on poisoning of humans and non-laboratory animals confirmed by detection of AC or its metabolites are available, although rarely on domestic animals. Furthermore, reports on clinical cases in domestic animals rarely report quantifications of AC in blood or body tissues. The present study describes the validation of a quantitative UHPLC-MS/MS method that can be used in cases of suspected AC poisoning in cats. The validation study showed the method to be fit for purpose. In serum the limit of quantification was 100 ng/mL and the limit of detection 30 ng/mL. The new analytical method was applied on blood samples collected from 20 individual cats with a preliminary clinical diagnosis of acute AC poisoning. Alpha-chloralose was confirmed in all 20 feline blood samples, and the concentration range of AC was 538 -17 500 ng/mL. The quantitative method developed in this study was found to be a fast and selective method for confirmation of AC poisoning using blood samples from cats.