Technical Abstract: In previous study, we have reported the development of simple algorithms for determining fiber maturity and crystallinity from Fourier transform (FT) -mid-infrared (IR) measurement. Due to its micro-sampling feature, we were able to assess the fiber maturity and crystallinity at different portions of a single cotton boll, as demonstrated here. Meanwhile, we attempted to compare the fiber maturity and crystallinity information between FT-mid-IR and X-ray diffraction (XRD) measurement. Despite considerable efforts in developing the curve-fitting protocol to evaluate the crystallinity index (CI) from XRD, in its present state XRD measurement can only provide a qualitative or semi-quantitative assessment of the amounts of crystalline or amorphous portion in a sample. The greatest barrier to establish quantitative XRD is the lack of appropriate cellulose standards needed to calibrate the measurements. In practice, samples with known CI are very difficult to be prepared or determined. As an approach, we might assign the samples with known CIs from FT-mid-IR procedure. Hence, this study also reported the development of a simple XRD algorithm, over time-consuming and subjective curve-fitting process, for direct determination of cotton cellulose CI by calibrating XRD with the use of CIIR as references.