Carbon-based Dots (CDs) and their functionalized (nano)composites have recently attracted a lot of attention, owing to their seemingly easy preparation and their numerous potential applications, ranging from biomedical (imaging and drug delivery) to (opto)electronics (i.e., solar cells and LEDs). This protocol covers the synthesis, purification and functionalization of nitrogen-doped carbon nanodots (NCNDs). Firstly, we describe the bottom-up synthesis of NCNCDs by using molecular precursors (arginine and ethylene diamine), through a microwave hydrothermal procedure. We also provide guidelines for the purification of these materials, either through filtration, dialysis or low-pressure size-exclusion chromatography. Secondly, we outline postfunctionalization procedures for the surface modification of NCNDs (such as alkylation and amidation reactions). Thirdly, we provide detailed instructions on the preparation of NCNDs with different properties (such as color emission, electrochemistry and chirality). Given the fast evolution in the preparation and application of CDs, issues that might arise from artifacts, errors and impurities should be avoided. In this context, the present protocol aims at helping synthesize high quality nanomaterials with high reproducibility, for various applications. Furthermore, even if CDs are prepared by different synthetic procedures, as well as from different molecular precursors, they can still be purified and characterized by following this protocol. The sample preparation takes various time frames, ranging from 2 to 12 days depending on the adopted synthesis and purification steps.