Cardiolipin (CL), is a unique class of membrane phospholipids that is found almost exclusively in the mitochondrial inner membrane of eukaryotic cells. CL undergoes substantial remodelling through a highly conserved deacylation-transacylation pathway, and Barth syndrome is caused by a deficiency of the CL transacylase, Tafazzin (TAZ). Because of their unique structure, conducting an in-depth examination of the CL molecular diversity is challenging from an analytical perspective. However, when travelling wave ion mobility is combined with high-resolution tandem mass spectrometry (IM-MS/MS), a rapid and simple multidimensional characterization of the CL molecular diversity is made feasible due to CL separation from obscuring singly charged ion signals. Calculating the peak intensities that overlap the monoisotopic CL ions has been made easier thanks to the Python script used to automate the procedure. All of the IMMS/MS-related parameters have been tuned to ensure CL separation and get optimum intensity, fragmentation, and quantitation from the samples. We demonstrate using IM-MS/MS that mutants of Saccharomyces cerevisiae lacking in CL remodelling (Cld1ΔTaz1Δ) exhibit a shift towards CL species with more saturated and shorter acyl chains. Furthermore, Saccharomyces cerevisiae mutant Taz1Δ also display a shift towards CL species with more saturated and shorter acyl chains but, the most relevant finding was that the MLCL/CL ratio has increased in a manner that is comparable to that seen in individuals with Barth syndrome. Our findings provide additional evidence that the CL profiles of different mammalian tissues are distinct from one another. In conclusion, IM-MS/MS is a method that is a powerful, robust and accurate tool when it comes to isolating CL species from complicated lipid samples and for identifying CL structural diversity.