The Scale of Positive and Negative Experience (SPANE) is a self-report questionnaire designed to assess positive and negative emotions. In Study 1 (N = 4,250, 61.95% females, M-age = 28.56 years), we evaluated measurement invariance of the SPANE across gender and age using moderated factor analysis. In Study 2 (N = 200, 52.5% females, M-age = 21.82 years), we investigated the convergent and discriminant validity of the SPANE by examining its associations with measures of well-being and religiosity. In Study 3 (N = 160, 87.5% females, M-age = 20.38 years), we used a prospective design to examine associations of the SPANE with the Big Five personality traits. The results provided general support for the measurement invariance of the SPANE across age and gender, but some non-invariant items were detected as well. The analyses of latent mean differences across gender revealed that women reported higher levels of both positive and negative emotions than men, but the effect size for positive emotions was very small. Older participants reported lower levels of positive emotions and higher levels of negative emotions than younger participants. Both convergent and discriminant validity of the SPANE were supported.