Spinach is one of edible leafy vegetable in many countries worldwide and is the most prime and informative plant species for studying plant chloroplast. A main advantage of spinach which makes it remarkable among other plants is that its leaf possesses abundant number of chloroplasts and each chloroplast sustains a very large genomic molecule. This characteristic of spinach makes it a unique crop that can serve as a valuable genetic model for studying chloroplast manipulation at the molecular genetic level. Therefore, implementing a technique which can lead to identify an appropriate way to induce adequate plant regeneration from explants has become significantly important with a great demand. A study was conducted to examine optimization of callogenesis and regeneration of two spinach cultivars; Orai and Viroflay using MS based medium with various combination of plant growth regulators (PGRs); IAA, 2,4-D and GA3 at different concentration rates in order to evaluate regeneration induction of leaf, hypocotyl and cotyledon explants under three separate experimental studies. The results indicated that leaf explants from both cultivars were capable of producing the highest callogenesis on growth media which supplemented with 0.5 mg/l 2,4-D and 2 mg/l Kinetin. When calli was transferred into medium containing concentration of 0.1, 2 and 1 mg/l of 2,4-D, Kinetin and GA3 respectively, only the explants of Orai cultivar was able to induce regeneration. Also, hypocoyl drived calli from Viroflay cultivar showed the best regeneration when transferred on medium containing 2 mg/l IAA and 3.4 mg/l GA3. There was no regeneration from cotyledon explants of both cultivars on all media.