BackgroundGame-based learning (GBL) is effective for increasing participation, creativity, and student motivation. However, the discriminative value of GBL for knowledge acquisition has not yet been proven. The aim of this study is to assess the value of Kahoot! as a discriminative tool for formative assessment in medical education in two different subjects.MethodsA prospective experimental study was conducted on a sample of 173 students enrolled in neuroanatomy (2021–2022). One hundred twenty-five students individually completed the Kahoot! prior to the final exam. In addition, students enrolled in human histology during two academic courses were included in the study. The control group course (2018–2019) received a traditional teaching methodology (N = 211), while Kahoot! was implemented during 2020–2021 (N = 200). All students completed similar final exams for neuroanatomy and human histology based on theory tests and image exams.ResultsThe correlation between the Kahoot score and the final grade was analyzed for all students enrolled in neuroanatomy who completed both exercises. The correlation between the Kahoot exercise and the theory test, image exam and final grade was significantly positive in all cases (r = 0.334 p < 0.001, r = 0.278 p = 0.002 and r = 0.355 p < 0.001, respectively). Moreover, students who completed the Kahoot! exercise obtained significantly higher grades in all parts of the exam. Regarding human histology, the theory tests, image exams and final grades were significantly higher when using Kahoot! versus the “traditional” methodology (p < 0.001, p < 0.001 and p = 0.014, respectively).ConclusionsOur study demonstrates for the first time that Kahoot! can be used to improve and predict the final grade in medical education subjects.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12909-023-04379-x.