1H-15N HSQC spectra analyzed by CHESPA and CHESCA should be acquired using 15N uniformly labelled protein samples. If the protein sample is bound to a ligand, the protein needs to be fully saturated. We recommend that, for each bound state, HSQC spectra are acquired with at least three different ligand concentrations to ensure saturation. All the HSQC spectra should be properly referenced before the chemical shift perturbation analysis. A less accurate method is to use the residual H2O signal and the 15N to 1H γ ratio to reference the spectra. However, it is recommended to use a referencing compound instead of the aforementioned method. We used 15N-acetyl-glycine as the referencing compound in this article to reference all the spectra before CHESPA and CHESCA. Before starting the analysis, all spectra should be properly assigned with 1-letter amino acid code and residue numbers (e.g. G1N-H). The residue numbers should be kept consistent among all spectra. Automated resonance assignments can be easily obtained from I-PINE web server using the PINE-SPARKY.2 program equipped in the POKY software package (two-letter-code ep). One can transfer the assigned peaks from one spectrum to another by copy and paste ornaments (control + a and control + c in the assigned spectrum, and control + v to the target spectrum)