The seasonal production of Allium cepa var. ascalonicum causes a rise in its demand during the off-season. Consumers mostly prefer onion cultivars like the 'Super Philip', because of their high productivity, large and round bulbs, shiny appearance, and less spicy taste. In plant breeding, polyploidy induction through mutagens is a technique often used to produce shallot cultivars of better quality. Bio-Catharantin from the leaf extract of Catharanthus roseus L. is used as a polyploid induction agent instead of colchicine. The latest study aimed to determine the effect of Bio-Catharantin concentration (0.2% and 0.4%) on phenotypic traits (plant height, bulb mass, and the number of bulbs), and the chromosome number to determine the minimum concentration that could cause polyploidization in shallots. The research was conducted from December 2020 to February 2021 in a greenhouse in Madurejo, Prambanan, and the Laboratory of Genetics and Breeding, Faculty of Biology, Gadjah Mada University, Indonesia. Bio-Catharantin concentration did not affect plant height which was comparable with the control. Both treatments caused an increase in bulb mass up to 37.7 and 41.76 g at the concentrations of 0.2% and 0.4%, respectively, compared with the control (31.47 g). The number of bulbs increased up to 10.6 and 9.8 g for 0.2% and 0.4% concentrations, respectively, compared with 8.8 in the control. The ploidy level of cells was increased from 2n (16) to 3n (24) at 2% and 4n (32) at 4% Bio-Catharantin.