AbstractMotor learning and flexibility allow animals to perform routine actions efficiently while keeping them flexible. There is a number of paradigms used to test cognitive flexibility but not many of them focus specifically on learning of complex motor sequences and their flexibility. While many tests use operant or touchscreen boxes that offer high throughput and reproducibility, the motor actions themselves are mostly simple presses of a designated lever. To focus more on motor actions during the operant task and to probe the flexibility of these well-trained actions, we developed a new operant paradigm for mice, the, “timed sequence task”. The task requires mice to learn a sequence of lever presses that have to be emitted in precisely defined time limits. After training, the required pressing sequence and/or timing of individual presses is modified to test the ability of mice to alter their previously trained motor actions. We provide a code for the new protocol that can be used and adapted to common types of operant boxes. In addition, we provide a set of scripts that allow automatic extraction and analysis of numerous parameters recorded during each session. We demonstrate that the analysis of multiple performance parameters is necessary for detailed insight into animals’ behavior during the task. We validate our paradigm in an experiment using the valproate model of autism as a model of cognitive inflexibility. We show that the valproate mice show superior performance at specific stages of the task, paradoxically due to their propensity to more stereotypic behavior.Significance StatementCognitive flexibility impairment is a crucial component of many neurological disorders and it is frequently evaluated in animal models. As the commonly used tests usually do not focus on motor learning and the ability to adapt motor sequences, we designed a new paradigm to evaluate motor learning and its flexibility. The timed sequence task is automatized and easily accessible as it is based on widely available operant boxes. During the training, the task requires precise timing of each action to force stereotypic performance. Its relative complexity allows detailed analysis of multiple parameters and therefore detailed insight into animal’s behavior. The task can be used to reveal and understand subtle differences in motor and operant learning and flexibility.Graphical Abstract