Background: There is increasing recognition that supermarkets are an important environment for health-promoting interventions such as fiscal food policies or front-of-pack nutrition labeling. However, due to the complexities of undertaking such research in the real world, well-designed randomized controlled trials on these kinds of interventions are lacking. The Virtual Supermarket is a 3-dimensional computerized research environment designed to enable experimental studies in a supermarket setting without the complexity or costs normally associated with undertaking such research. Objective: The primary objective was to validate the Virtual Supermarket by comparing virtual and real-life food purchasing behavior. A secondary objective was to obtain participant feedback on perceived sense of "presence" (the subjective experience of being in one place or environment even if physically located in another) in the Virtual Supermarket. Methods: Eligible main household shoppers (New Zealand adults aged ≥18 years) were asked to conduct 3 shopping occasions in the Virtual Supermarket over 3 consecutive weeks, complete the validated Presence Questionnaire Items Stems, and collect their real supermarket grocery till receipts for that same period. Proportional expenditure (NZ$) and the proportion of products purchased over 18 major food groups were compared between the virtual and real supermarkets. Data were analyzed using repeated measures mixed models. Results: A total of 123 participants consented to take part in the study. In total, 69.9% (86/123) completed 1 shop in the Virtual Supermarket, 64.2% (79/123) completed 2 shops, 60.2% (74/123) completed 3 shops, and 48.8% (60/123) returned their real supermarket till receipts. The 4 food groups with the highest relative expenditures were the same for the virtual and real supermarkets: fresh fruit and vegetables (virtual estimate: 14.3%; real: 17.4%), bread and bakery (virtual: 10.0%; real: 8.2%), dairy (virtual: 19.1%; real: 12.6%), and meat and fish (virtual: 16.5%; real: 16.8%). Significant differences in proportional expenditures were observed for 6 food groups, with largest differences (virtual - real) for dairy (in expenditure 6.5%, P<.001; in items 2.2%, P=.04) and fresh fruit and vegetables (in expenditure: -3.1%, P=.04; in items: 5.9%, P=.002). There was no trend of overspending in the Virtual Supermarket and participants experienced a medium-to-high presence (88%, 73/83 scored medium; 8%, 7/83 scored high). Conclusions: Shopping patterns in the Virtual Supermarket were comparable to those in real life. Overall, the Virtual Supermarket is a valid tool to measure food purchasing behavior. Nevertheless, it is important to improve the functionality of some food categories, in particular fruit and vegetables and dairy. The results of this validation will assist in making further improvements to the software and with optimization of the internal and external validity of this innovative methodology.