Kinematic analysis of the digits using optical motion capture systems relies on defining accurate coordinate systems for the individual segments. Limitations of previous digit kinematic protocols include marker placement errors, marker occlusion and superimposition, and skin movement artifact. The purpose of this study was to develop a protocol utilizing a digit alignment device (DAD) and nail marker clusters to overcome these limitations. Ten subjects underwent 10 static calibration trials for validation. The orientation of the thumb distal phalange relative to the index finger distal phalange was described using Euler angles of pitch(x), yaw(y'), and roll(z''). The digit calibration protocol demonstrated high accuracy (0.5°, 1.9° and 2.2° for x, y', z'') and precision (1.4°, 2.3° and 3.1° for x, y', z''). The developed protocol provided convenient identification of transformations that determine anatomically relevant coordinate systems for the distal phalanges of the digits. The potential of utilizing this protocol as a standardized tool for digit kinematics was demonstrated using a dynamic task of precision pinching.