Much progress has been made in digital microfluidic biochips (DMFB), with a great body of literature addressing low-cost, high-performance, and reliable operation. Despite this progress, security of DMFBs has not been adequately addressed. We present an analysis of a DMFB system prone to malicious modification of routes and propose a DMFB defense based on spatio-Temporal randomized checkpoints using CCD cameras. Absent the knowledge of the time-and space-randomized checkpoints, an attacker cannot navigate the DMFB without alerting the system. We present an algorithm to guide the placement and timing of the checkpoints such that the probability that an attack can evade detection is minimized. The efficacy of the defense mechanism is illustrated with a case study under stealthy malicious modifications.