Peer Instruction (PI) is an instructional strategy for engaging students during class through a structured questioning process that improves the learning of the concepts of fundamental sciences. Although all students are supposedly engaged in discussions with their peers during Peer Instruction, the learning gains generally remain at a medium level, suggesting a lack of participation of certain students who do not benefit from social interactions. The present study examined whether the Stepladder technique might optimize the Peer Instruction method and increase learning gains. With this technique, students enter a group sequentially, forcing every group member to participate in discussions. Eighty-four chemistry students were asked to answer easy and difficult multiple-choice questions before and after being randomly assigned to one of three instructional conditions during a chromatography lesson (Classic PI vs. Stepladder PI vs. Individual Instruction without any discussion with peers). As predicted, results showed that learning gains were greatest in the Stepladder PI group, and that this effect was mainly observed for difficult questions. Results also revealed higher perceived satisfaction when students had to discuss the questions with their peers than when they were not given this possibility. By extending the Stepladder technique to higher education, these findings offer a step forward in the Peer Instruction literature, showing how it can enhance learning gains.