There has been a rapid increase in demand for premium and safe agricultural products. Protected systems, such as greenhouses, are being adopted to meet demand. Ease in environmental regulation required for optimal plant growth is one of the advantages of protected systems. However, drawbacks such as poor ventilation in greenhouses can be fatal to the human workforce. This has led to the development of robots for hazardous tasks. Considering mobile robots are required to navigate down every aisle to perform a task in a greenhouse, and it is difficult to predict at which point the robot will need to return to the start point, to offload or refill for transportation and spraying schedules, respectively or battery charges. It will be commercially constraining to manufacture robots for every greenhouse specification. Efficient navigation can be done through path planning or layout design. In this paper, the greenhouse layout optimization problem was formulated to find optimal points on each bed to create an access path that would enable a reduction in the total travel time from all points in the greenhouse to the base point. The optimization problem was solved using differential evolution (DE), an evolutionary algorithm. Furthermore, we considered: 1) required space for inter-bed and rotary robot navigation; 2) standard bed specification; 3) area of the greenhouse; and 4) base point for starting and terminating navigation. The applicability of the proposed method was demonstrated by carrying out the experimental simulations on several greenhouse sizes.