We present a facile method for the production of fluorescent diamond nanocrystals (DNCs) of different sizes and efficiently quantify the concentration of emitting defect color centers (DCCs) of each DNC size. We prepared the DNCs by ball-milling commercially available micrometer-sized synthetic (high pressure, high temperature (HPHT)) diamonds and then separated the as-produced DNCs by density gradient ultracentrifugation (DGU) into size-controlled fractions. A protocol to enhance the uniformity of the nitrogen-vacancy (NV) centers in the diamonds was devised by depositing the DNCs as a dense monolayer on amino-silanized silicon substrates and then subjecting the monolayer to He(+) beam irradiation. Using a standard confocal setup, we analyzed the average number of NV centers per crystal, and obtained a quantitative relationship between the DNC particle size and the NV number per crystal. This relationship was in good agreement with results from previous studies that used more elaborate setups. Our findings suggest that nanocrystal size separation by DGU may be used to control the number of defects per nanocrystal. The efficient approaches described herein to control and quantify DCCs are valuable to researchers as they explore applications for color centers and new strategies to create them.