Sir,
When the RMSE appears on the screen, what is it's unit?Is it in a.u. as mentioned in page 277
https://i.postimg.cc/kDpF2rQt/Screenshot-20181022-154732.png

When no unit is explicitly given in the output, a.u. is used as default. Therefore, the RMSE of ESP is reported in a.u.

Thank you Sir for correcting me. I will upload the C2H2.wfn file. However, I was wondering if there is a way out to specify the Merz-Kollman grid spacing points in calculation of the electrostatic potential. Any suggestion or comment is highly appreciated.

For MK grids, there is no so-called "grid spacing", because the grids are not evenly placed around the molecule. You can only control density of points per Angstrom^2 on MK layers.

Sorry Sir for the incomplete msg
I wanted to use my own specified grid setting and then calculate the RMSE of the molecule
For this I used the C2H2.wfn file. These are the steps I followed
1. Went to main function 5 (Output and plot specific property within a spatial region)
2. Then went available real space function option 12( Total electrostatic potential
...

You steps are incorrect, and the result is meaningless.

You do not need to manually generate cube file of ESP, therefore the steps 1~6 are completely redundant. Also, to examine the ESP reproducibility of given atomic charges by RMSE, the input file that loaded when Multiwfn boots up must contain wavefunction information (so that ESP can be evaluated based on wavefunction), for example you can use .wfn/.wfx/.fch and so on, while .cube file should not be used since it doesn't contain wavefunction information.

Therefore, you should delete steps 1 to 7, and in step 8 you should load C2H2.wfn. If you are still confused, I suggest you first reproduce the example in Section 4.7.8 of the latest version of manual.

Sir,
The procedure that I used to change the grid setting for my input file is as follows. Is it the correct way?
1. Loading the wfn file
2. Going to mode 5 setting the grid
3. Calculating the esp and changing the Aug3D for grid extension to 5 angstron beyond the molecule.
4. Changing the grid spacing in mode 6
5. Exporting the output to the cube file
6. Booting up Multiwfn again and loading the cube file with the grid setting
7. Calculating the atomic charge RMSE by entering the MK module
8. Changing the layer scale factor according to my specifications since I want only to consider those grid points lying in between a specified range.
The output is generated. So, can I assume I am doing correctly?

I am totally confused, what do the mode 5 and mode 6 you mean? Since there are numerous options in Multiwfn, if you do not provide full operation steps to me, I really feel difficult to reply your question. (You can copy full output from Multiwfn window to a .txt file and upload it)

If you feel that specifying grid spacing every time is too troublesome, you can use silent mode to run Multiwfn, see section 5.2 of the manual, you can add the commands used to setting grid spacing in your inputstream file, so that you do not need to manually input the command in each time of calculation.