Protein secretion is a complex, multistep process that relies on signal sequences to guide recombinant proteins through various stages of the secretion pathway, ultimately targeting these proteins to the extracellular matrix. The mating factor alpha (MFα) signal peptide, initially identified for its role in the mating factor protein export in Saccharomyces cerevisiae, has emerged as the most effective tool for the secretory production of recombinant proteins in yeast expression systems, including Komagataella phaffii (reclassified from Pichia pastoris). Over the years, numerous studies have aimed at enhancing the efficiency and processability of the MFα signal sequence, resulting in several optimized variants. In this chapter, we highlight the most frequently used and highly effective MFα signal leader variants. We present a streamlined and universal cloning strategy using pPpT4_MFα as a vector backbone and the gene encoding eGFP as the gene of interest to facilitate the use of these variants for recombinant protein secretion. This step-by-step approach also provides the precise (codon optimized) nucleotide and amino acid sequences of these MFα signal leader variants.