Abstract In Malaysia, the escalating annual road traffic accidents, primarily attributed to human factors, necessitate a closer examination of these influences. This study focuses on assessing the impact of lane segregation on motorcyclists’ behavior within multilane roadways. Conducted along the R5 highway from Kapar to Kuala Selangor, classified as a rural highway, the study evaluates the 85th percentile operating speed, crucial for multilane flow rate. Two locations, one with median physical barriers and the other with only line markings, were compared. Traffic flow and speed were observed at 5-minute intervals over 45 minutes using a laser gun and tally counter. Analysis, based on the Malaysian Highway Capacity Manual, revealed that the median barrier location promoted safe speeds and a higher motorcycle percentage. Conversely, the absence of a median barrier but with line markings resulted in elevated speeds exceeding the limit, accompanied by a lower motorcycle percentage. These findings underscore the significance of median physical barriers in reducing vehicle speed, enhancing road safety, and curbing road accidents.