Identifying optimal reaction coordinates for complex conformational changes and protein folding remains an outstanding challenge. This study combines collective variable (CV) discovery based on chemical intuition and machine learning with enhanced sampling to converge the folding free energy landscape of lasso peptides, a unique class of natural products with knot-like tertiary structures. This knotted scaffold imparts remarkable stability, making lasso peptides resistant to proteolytic degradation, thermal denaturation, and extreme pH conditions. Although their direct synthesis would enable therapeutic design, it has not yet been possible due to the improbable occurrence of spontaneous lasso folding. Thus, simulations characterizing the folding propensity are needed to identify strategies for increasing access to the lasso architecture by stabilizing the pre-lasso ensemble before isopeptide bond formation. Herein, harmonic linear discriminant analysis (HLDA) is combined with metadynamics-enhanced sampling to discover CVs capable of distinguishing the pre-lasso fold and converging the folding propensity. Intuitive CVs are compared to iterative rounds of HLDA to identify CVs that not only accomplish these goals for one lasso peptide but also seem to be transferable to others, establishing a protocol for the identification of folding reaction coordinates for lasso peptides.