Squalene is a naturally occurring triterpene widely utilized in the pharmaceutical and cosmetic industries due to its strong antioxidant and anticancer properties. Pichia pastoris, a methylotrophic yeast, has emerged as a promising host for the biosynthesis of high-value compounds because of its high protein expression capacity, robust growth performance, and ease of genetic manipulation. The present study investigates strategies to enhance squalene production in the P. pastoris X-33 strain through metabolic engineering and process optimization approaches. To improve squalene biosynthesis, pathway engineering was performed by targeting key genes, including tHMG, ERG9, and ERG20. The individual effects of these genes on squalene yield were evaluated by overexpressing those encoding rate-limiting enzymes within the biosynthetic pathway. Furthermore, shake-flask fermentation conditions were optimized to increase metabolic flux toward squalene production.