This study investigated the effect of differentiated instruction on Turkish preservice teachers' academic achievement and self-directed learning readiness in an online teaching profession course. The study utilized a pretest-posttest control group design. Data were collected through an achievement test and a self-directed learning readiness scale. The independent samples t-test, paired samples t-test, and analysis of covariance were used to analyze the data. The findings revealed that online differentiated instruction had a significant impact on the academic achievement of preservice teachers, while it did not create a significant difference in terms of total self-directed learning readiness, self-management, desire for learning, or self-control. As a result, it is seen as essential for teacher educators to conduct differentiated instruction in order to enhance preservice teachers' academic achievement and motivate them to use this approach in their future classes. It is also suggested that teacher educators provide preservice teachers with sufficient support to improve their self-directed learning readiness.