A General Defocusing Particle Tracking (GDPT) method is proposed for tracking the three-dimensional motion of particles in Lab-on-a-chip systems based on a set of calibration images and the normalized cross-correlation function. In comparison with other single-camera defocusing particle-tracking techniques, GDPT possesses a series of key advantages: it is applicable to particle images of arbitrary shapes, it is intuitive and easy to use, it can be used without advanced knowledge of optics and velocimetry theory, it is robust against outliers and overlapping particle images, and it requires only equipment which is standard in microfluidic laboratories. We demonstrate the method by tracking the three-dimensional motion of 2 μm spherical particles in a microfluidic channel using three different optical arrangements. The position of the particles was measured with an estimated uncertainty of 0.1 μm in the in-plane direction and 2 μm in the depth direction for a measurement volume of 1510 × 1270 × 160 μm(3). A ready-to-use GUI implementation of the method can be acquired on .