Cystine-knot peptides, with their exceptional thermal stability and resistance to proteolytic degradation, present highly favorable biophysical properties for use as scaffolds in engineering of binders for diverse applications. Multiple variants of knottins with prescribed functional characteristics were described in the literature. These were obtained by rational engineering of natural peptides or by combinatorial library screening. Herein, we describe in detail an ultra-high-throughput screening method of cystine-rich peptide libraries making use of yeast surface display. This is exemplified by converting a trypsin-like protease inhibiting oMCoTI-II cystine-knot framework to a binder of the immune-oncology target CTLA-4. Combinatorial library cloning, followed by yeast surface presentation and sorting, analysis of single clones, and finally chemical synthesis of cystine-knot peptides and their functional validation, provides a robust method for obtaining cystine-rich peptides with novel functional characteristics.