Hemoglobin (Hb) variants arise due to point mutations in globin chains and their pathological treatments rely heavily on the identification of the nature and location of the mutation in the globin chains. Traditional methods for diagnosis such as HPLC and electrophoresis have their own limitations. Therefore, the present study aims to develop and optimize a specific method of sample processing that could lead to improved sequence coverage and analysis of Hb variants by nano LC-MALDI MS/MS. In our study, we primarily standardized various sample processing methods such as conventional digestion with trypsin followed by 10% acetonitrile treatment, digestion with multiple proteases like trypsin, Glu-C, Lys-C, and trypsin digestion subsequent to 2,2,2 trifluoroethanol (TFE) treatment. Finally, the peptides were identified by LC-MALDI MS/MS. All of these sample processing steps were primarily tested with recombinant Hb samples. After initial optimization, we found that the TFE method was the most suitable one and the efficiency of this method was applied in Hb variant identification based on high sequence coverage. We developed and optimized a method using an organic solvent TFE and heat denaturation prior to digestion, resulting in 100% sequence coverage in the β-chains and 95% sequence coverage in the α-chains, which further helped in the identification of Hb mutations. A Hb variant protein sequence database was created to specify the search and reduce the search time. All of the mutations were identified using a bottom-up non-target approach. Therefore, a sensitive, robust and reproducible method was developed to identify single substitution mutations in the Hb variants from the sequence of the entire globin chains. Over 330,000 infants are born annually with hemoglobinopathies and it is the major cause of morbidity and mortality in early childhood. Hb variants generally arise due to point mutation in the globin chains. There is high sequence homology between normal Hb and Hb variant chains. Due to this high homology between the two forms, identification of variants by mass spectrometry is very difficult and requires the full sequence coverage of α- and β-chains. As such, there is a need for a suitable method that provides 100% sequence coverage of globin chains for variant analysis by mass spectrometry. Our study provides a simple, robust, and reproducible method that is suitable for LC-MALDI and provides nearly complete sequence coverage in the globin chains. This method may be used in the near future in routine diagnosis for Hb variant analysis.