Several antibody fragment engineering techniques aim at intrinsic stability enhancement, but are not applied in a truly generic way. Here, a strategy is proposed whereby consistent gain in stability is accomplished by introducing a specific disulfide bond between two opposite beta-strands in the hydrophobic core of the immunoglobulin heavy-chain variable domain of heavy-chain antibodies (Nanobody). Besides the rational design of a disulfide bond between residues 39 and 87, a Nanobody harboring an extra naturally occurring cystine between residues 54 and 78 was compared to an equivalent Nanobody without that cystine. Both novel disulfide cross-links were introduced in several Nanobodies in various combinations. Interestingly, only the extra naturally occurring cystine consistently increased the conformational and thermal stabilities of wild-type Nanobodies without affecting antigen binding.