The problems of new psychoactive substances (NPSs), especially related to drivers, constitute an open research area. In this case report, we present a traffic accident case, in which two passengers of five individuals died instantly, while the other three persons survived the accident with minor injuries only. From the blood samples of the driver and the passengers, α-pyrrolidinovalerophenone (α-PVP), an NPS belonging to the category of cathinone derivatives, was disclosed. Therefore, we established a detailed procedure for analysis of α-PVP in blood samples by liquid chromatography-tandem mass spectrometry. After careful validation tests of this method, α-PVP concentration in blood samples from the surviving driver and passengers, and from the two deceased, were measured. The concentrations varied from 20 to 650 ng/mL. Access to detailed information originating from the court files and from explanations provided by the driver and eye witnesses revealed extremely valuable illustrative details addressing the symptoms and pharmacological effects of α-PVP on the human organism, thus contributing to enriching the body of knowledge of α-PVP abuse.