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Beetroot betalains (E162) as natural colorants, extracted by physical means, are permitted by the European Union for use as a natural colorant in foods. Like other natural colorants, betalains have limitations due to the lack of standardized extraction protocols and reduced stability. An experiment was conducted for the process optimization of betalains extraction from a competent variety and subsequently its stabilization through acidulants. Results revealed that the Crimson Globe cultivar was a feasible matrix for betalains extraction over Detroit Dark Red. Box Behnken Design of Response Surface Methodology (RSM) optimized that at 70°C, 4.0 min extraction time and 1:3 solid to solvent ratio, maximum betalains and phenolics could be extracted. Betalains were found to be more related to color expression (r =.962) than the antioxidant activity (r =.688). Among various acidulants, betalains was maximally retained by 1% (w/v) ascorbic acid with t1/2 = 155.17 h followed by citric (92.36 h), tartaric (85.20 h), succinic (83.96 h), and malic acid (75.72 h). Novelty impact statement: Beetroot betalains, designated as GRAS, are known for their antioxidant and coloring potential. Detailed extraction methodology for betalains was optimized with pure aqueous solvent so that the extracted betalains, when used as a food colorant are devoid of any solvent residue. Further, the study concluded that among various acidulants, the performance of ascorbic acid was best in terms of better color and antioxidant stability. The betalains acidified with ascorbic acid could be stored up to 9 months at 10°C. This is the first detailed report on aqueous extraction protocol standardization from beetroot cv. Crimson Globe and its stabilization using acidulant/s. [ABSTRACT FROM AUTHOR]