Precision fermentation is a promising route to develop recombinant proteins with improved functionality. Protein functionality is highly influenced by protein structure, which can be significantly affected by conformational restraints induced by cysteines. Here, we study the impact of individual cysteines on β-lactoglobulin structure, by stepwise substituting them with alanine. Structural characterisation was performed from secondary to quaternary level. The findings demonstrated that the free Cys121 played no role in the folding and dimerisation of BLG. The β-barrel usually includes one disulphide bond (Cys106–Cys119), which was found to be crucial for its formation. The usually exposed disulphide bond (Cys66–Cys160) played no major role in folding of the β-barrel, but mainly modulated its accessibility and mediated dimerisation. Thereby, cysteine mutations can be used as a tool to modify protein structures. These different structures can now be used to further understand the impact on protein behaviour and their functionality.