Aqueous magnetic nanofluid consists of superparamagnetic nanoparticles, with a typical size of 10–12 nm. On the application of a magnetic field, these nanoparticles align heterogeneously and form a chain or chain-like structure. This structure is observed using a microscope. Although many articles report such chain or microstructure formation well, the method to identify and determine chain parameters, e.g., chain length, width, and associated counts, is scarce. Similarly, interchain or successive distance is one of the critical parameters for developing magnetic nanofluid-based devices. The work describes magnetic field-induced chain parameters (MFCP) and magnetic field-induced interchain distance (MFID), a set of developed protocols in the ImageJ software to identify and determine the (i) chain length, width, and associated counts, along with (ii) successive distance of the magnetic chains in the magnetic nanofluid. This utilizes a macro file such as MFCPji.txt and MFIDji.txt for ImageJ, which can be used on microscopic images of magnetic nanofluids without applying a magnetic field. The protocol does not require specialized scientific equipment and can be carried out using open-source software ImageJ/Fiji. The examples of microstructure formations in two different magnetic fluids (A and B) are discussed. In addition, the results of the associated weighted average chain length, chain width, number of chains, and the successive chain distance are reported. The chain parameters are helpful to determine diffraction grating angles. The MFCPji and MFIDji macros have been integrated into a macro toolset that can be configured to be run on ImageJ startup. The MFCPji and MFIDji are available from the following Uniform Resource Locator (URLs): https://github.com/urveshsoni/ImageJ-Macros and https://ruchadesailab.wordpress.com/publication/