Two pairs of alignment targets (one aligned, one misaligned by a bias distance) are formed on different masks to produce a first pair of conjugated interference patterns. Other pairs of alignment targets are also formed on the masks to produce a second pair of conjugated interference patterns that are inverted the first. Misalignment of the dark and light regions of the first interference patterns and the second interference patterns in both pairs of conjugated interference patterns is determined when patterns formed using the masks are overlaid. A magnification factor (of the interference pattern misalignment to the target misalignment) is calculated as a ratio of the difference of misalignment of the relatively dark and relatively light regions in the pairs of interference patterns, over twice the bias distance. The interference pattern misalignment is divided by the magnification factor to produce a self-referenced and self-calibrated target misalignment amount, which is then output.