The purpose of this study was to assess the reliability and validity of the Spanish version of the Health Promoting Lifestyle Profile II (HPLP-II) scale in Colombian university students. This was a methodological study to verify reliability and construct validity. A total of 763 undergraduate university students in Cali, Colombia, agreed to participate in the study by filling out a form that included information on sociodemographic characteristics and the HPLP-II scale Spanish version. Data were collected between February and June 2021. To determine construct validity, a confirmatory factor analysis was performed, and internal consistency was determined through Cronbach's alpha. The confirmatory factor analysis of the proposed theoretical model showed that the goodness-of-fit indices of the scale demonstrated an acceptable level of validity nearing an excellent level of fit (χ2 = 7168.98; gl = 1268; p < 0.001; root mean square error of approximation = 0.08; normed fit index, adjusted goodness-of-fit index = 0.95). Cronbach's alpha coefficient of the scale was 0.94, and the subscales ranged from 0.68 to 0.89. The HPLP-II Spanish version is a valid and reliable instrument to assess the health-promoting lifestyle profile of university students.