There is evidence of the effectiveness of implementing mental health literacy (MHL) programs. However, there are substantial limitations in the instruments available for measuring MHL. This study aimed to develop and validate the EspaiJove.net MHL test (EMHL) for Spanish adolescents by assessing its psychometric properties. The development of the EMHL test was conducted using item pool generation and a pilot study. A convenience sample of students aged 13–15 years (n = 355) participated in the validity study. Reliability was assessed for internal consistency and via test-retest. Convergent validity was evaluated by comparing the effect sizes among known groups with different levels of mental health knowledge, the correlation with mental health-related instruments, and the item discrimination index. A final version of a 35-item EMHL test was obtained with two parts: (i) a binary choice format (yes/no) for the identification of mental disorders; (ii) a multiple choice question with four possible answer options. Internal consistency was acceptable in the first part (Cronbach’s alpha = 0.744; Guttman’s lambda 2 = 0.773) and almost acceptable in the second part (Cronbach’s alpha = 0.615; Guttman’s lambda 2 = 0.643). The test-retest evaluation supported the stability of the test (first part, ICC = 0.578; second part, ICC = 0.422). No ceiling and floor effects were found. The EMHL test scores discriminated between known groups with different levels of mental health knowledge and it is associated with several-related constructs of MHL. Conclusions: The EMHL test is a relevant measure for assessing MHL in adolescents into Spanish context with acceptable validity and stability.