Periwinkle Cathranthus roseus  is an ornamental plant that produces over 200 alkaloids, including vincristine, vinblastine, and vindoline, which are used to treat various types of cancer. However, the low yield of TIAs from periwinkle and the high cost of extraction has led researchers to explore alternative methods for their production. The study aims to investigate the influence of polyethylene glycol (PEG) as an elicitor to enhance alkaloid production in periwinkle callus cultures while examining the efficacy and safety of using PEG. Callus was induced and abiotic elicitation experiments were conducted to study callus growth enhancement. The dry weight of the callus was calculated, and vincristine and vinblastine were extracted and analyzed using high-performance liquid chromatography. The results showed that low to moderate concentrations of PEG4000 can enhance callus growth, while higher concentrations can reduce it. The addition of PEG4000 also increased the production of important alkaloids, including vindoline, vincristine, and vinblastine, by up to 2.65-fold. The findings suggest that PEG4000 can be used to optimize the production of alkaloids in C. roseus callus cultures.