It is important to quantify the amount of crossover innervation from the anterior interosseous nerve (AIN) through Martin-Gruber anastomosis (MGA) particularly in patients with high ulnar nerve injury who undergo nerve transfer surgery. The objective of this study is to describe a novel electrophysiological method for quantifying innervation from the AIN that can be done using conventional nerve conduction study setup and commonly available software for analysis. Seven subjects with MGA and nine patients who had undergone AIN to ulnar nerve transfer underwent conventional motor nerve conduction studies. Recording was done over the hypothenar and first dorsal interosseous muscles while stimulating the median and ulnar nerves at the wrist and elbow. Datapoint-by-datapoint subtraction of the compound muscle action potentials evoked at the elbow and wrist was performed after they had been onset-aligned. The results were compared to the collision technique and innervation ratio method. Results from the digital subtraction method were highly correlated with the collision technique (r= 0.96, p< 0.05). In contrast, its correlation with the innervation ratio method is substantially lower. In comparison to previously described techniques, the digital subtraction method has a number of practical advantages. It uses conventional nerve conduction study setup, and the added step of digital alignment and subtraction can be done through commonly available software. With the increasing use of nerve transfer surgery in severe high ulnar nerve injury, this could be a useful method to identify the presence of MGA prior to surgery and for evaluating nerve recovery following surgery.