(1) Background: Diener's Scale of Positive and Negative Experiences (SPANE) assesses the presence and intensity of positive and negative affects, since these are considered basic aspects of the study of well-being. This article studies its psychometric properties in the general Colombian population. (2) We conducted a cross-sectional study of a sample of 1255 Colombians and we used structural equation modeling to confirm the bifactor structure. Additionally, we studied invariance by gender, and convergent and concurrent validity. (3) We found acceptable fit indicators for the bifactor model (CFI = 0.889, RMSEA = 0.046, SRMR = 0.059) as well as for the convergent (CFI = 0.909, RMSEA = 0.050, SRMR = 0.063) and concurrent (CFI = 0.966, RMSEA = 0.036, SRMR = 0.041) validity models. We did not confirm total invariance across gender, although we found configural and metric invariance, so percentiles by sex were provided. (4) Conclusions: The SPANE is a valid and reliable measure to assess well-being among the Colombian population, although we alert researchers to the risk of comparing affectivity average scores between sexes.