Background: In health professionals, negative stereotypes toward older adulthood have been associated with the difficulty in recognizing pathological processes and the refusal to care for older patients because of assuming that communication with them will be uncomfortable and frustrating. For these reasons, research on stereotypes in these groups has acquired growing importance. The usual strategy to identify and evaluate agist stereotypes is to use scales and questionnaires. Although multiple scales are currently used, in Latin America, the Questionnaire for the Evaluation of Negative Stereotypes Toward Older Adulthood (Cuestionario de Estereotipos Negativos sobre la Vejez [CENVE]), developed in Spain, is widely used but without evidence of construct validity in our context. In addition, although in the original version, a factorial structure of 3 factors was found, in later studies, a unifactorial structure was obtained. Objective: The objective is to study the construct validity of the CENVE in a sample of Colombian health personnel to clarify its factorial structure and concurrent validity. Likewise, the measurement invariance according to gender and age was studied. Methods: A nonprobabilistic sample of 877 Colombian health professionals and intern health students was obtained. The data were collected online using the LimeSurvey tool. To study the factor structure of the CENVE, 2 confirmatory factor analysis (CFA) models were carried out, one to test a single factor and the other to test the 3-related-factor structure. The factor measurement reliability was evaluated with the composite reliability index (CRI) and the average variance extracted (AVE). The measurement invariance was studied according to gender (men and women) and age (emerging adults, 18-29 years old, and adults, 30 years old or older). Using a structural equation model, the relationship between age and the latent CENVE total score was studied to obtain evidence of concurrent validity, since studies indicate that the younger the age, the greater the number of stereotypes. Results: The 1-factor structure was confirmed. The reliability results indicated that both indices show adequate values. Likewise, the existence of a strong invariance in measurement by gender and age group was verified. After contrasting the means of the groups, the results showed that men show more negative stereotypes toward old age than women. Likewise, emerging adults also showed more stereotypes than adults. We also verified that age is inversely related to the latent score of the questionnaire, such that the younger the age, the greater the stereotype. These results are in agreement with those obtained by other authors. Conclusions: The CENVE shows good construct and concurrent validity, as well as good reliability, and it can be used to assess stereotypes toward older adulthood in Colombian health professionals and health sciences college students. This will allow us to better understand the effect of stereotypes on agism.