In recent times, digital microfluidic biochips have received an appreciable recognition as one of the most promising platforms for lab-on-a-chip attainment. Such a compound system can replace most of the laboratory experiments by controlling nano-litre or micro-litre volume of droplets and yield more accurate and faster results depending upon electrowetting on dielectric (EWOD) principle. Being aware of the fact about the progress of traditional square electrode biochips in the digital microfluidic realm, here in this paper, we present two-dimensional regular hexagonal digital microfluidic electrode (HDMFB) array. A hexagonal chip array offers numerous advantages over a square array like droplet movement, mixing operation, speed, etc. To cope with this new design technique care should be taken for fluidic constraints and electrode constraints to ensure safe droplet routing. Here, we propose an algorithm for efficient control pin assignment on the chip such that no droplet interference on the chip array occurs during an assay operation. Moreover, a multiplexed assay operation is performed by a scheduling algorithm, and the result is compared with a previous work conducted on the conventional square electrode array. Finally, a comparative study is done on the proposed architecture and the existing one on some relevant issues. © 2019 IEEE.