Background: Mental health problems and suicide ideation are common in adolescents. Early detection of these issues could prevent the escalation of mental health–related symptoms in the long term. Moreover, characterizing different profiles of prevalent symptoms in conjunction with emotional regulation strategies could guide the design of specific interventions. The use of web-based screening (WBS) tools has been regarded as a suitable strategy to timely detect symptomatology while improving the appeal, cost, timeliness, and reach of detection in young populations. However, the evidence regarding the accuracy of these approaches is not fully conclusive. Objective: The study aims (1) to examine the capability of a WBS to identify adolescents with psychiatric symptoms and suicidality and (2) to characterize the mental health profiles of a large sample of adolescents using WBS. Methods: A total of 1599 Latin American Spanish-speaking adolescents (mean age 15.56, SD 1.34 years), consisting of 47.3% (n=753) female, 98.5% Chilean (n=1570), and 1.5% Venezuelan (n=24) participants, responded to a mental health WBS. A randomized subsample of participants also responded to the Mini International Neuropsychiatric Interview for Children and Adolescents (MINI-KID). McNemar χ2 and receiver-operating characteristic curves tested the detection accuracy of WBS contrasted with the MINI-KID. Latent profile analyses explored the symptomatic and emotional regulation profiles of participants. Results: Both measures showed an adequate level of agreement (area under the curve per symptom domain ranging from 0.70 to 0.89); however, WBS yielded a higher prevalence than MINI-KID for all psychiatric symptoms, except suicide ideation and depression. Latent profile analyses yielded 4 profiles—one of them presented elevated psychopathological symptoms, constituting 11% of the sample (n=175). Rumination (odds ratio [OR] 130.15, 95% CI 51.75-439.89; P<.001), entrapment (OR 96.35, 95% CI 29.21-317.79; P<.001), and defeat (OR 156.79, 95% CI 50.45-487.23; P<.001) contributed significantly to the prediction of latent profile memberships, while cognitive reappraisal did not contribute to the prediction of any latent profile memberships, and expressive suppression was only associated to profile-2 membership. Conclusions: WBS is acceptable for the timely detection of adolescents at risk of mental health conditions. Findings from the symptomatic and emotional regulation profiles highlight the need for comprehensive assessments and differential interventions.