Current methods for determination of calcium content in human milk are based on standard laboratory experiments, which are time consuming and require specially trained personnel. Available instruments to accomplish these experiments are usually designed for a sample volume in the range of several milliliters. These approaches are not applicable for in-situ determination of calcium. In this thesis, an inexpensive and rapid technique for quantification of calcium in human milk is presented, using only 5 L of sample fluid for operation and determining results within 2-3 minutes. Principles of colorimetric chemical analytical methods are miniaturized to fit a specific microfluidic chip, fabricated based on standard dry film photoresist technology. Downscaling challenges such as fluid flow and mixing, are brought under control after implementation of phaseguides in the chip design and utilizing hydrogel concept, respectively. Future steps can be taken toward developing a compact device to replace the spectrometric instruments, making it an ideal analytical device for non-laboratory environments. This method can also be generalized as a new comprehensive technique for colorimetric assays in micrometer scale for other chemical substances.