Motivation is a central concept in self-determination theory (SDT). The Treatment Self-Regulation Questionnaire (TSRQ), which assesses motivation (autonomous, controlled, etc.), has been widely used. However, less is known about its applicability to samples such as college students, who may be at risk of having unhealthy behavior in many areas (including smoking, poor dietary habits, alcohol, or tobacco consumption). As this population is transitioning to adulthood, research is needed to understand motivation and changing health patterns. In addition, the lack of instruments for this population in Spain has made the measurement validation process a priority. The purpose of this psychometric study was to adapt the TSRQ to Spanish college students and to examine its structural and validity across four health domains. Two samples of Spanish college students (n = 347 and n = 244) agreed to participate in the study. Participants completed a booklet containing measures of motivation, well-being, general health, anxiety, depression, and lifestyle. CFA supported a five-dimensional structure in each domain. Reliability values were also adequate for each questionnaire. Regarding other sources of validity, statistically significant relationships between self-determination, health, and well-being were clearly confirmed, and autonomy was a significant predictor of lifestyle. The Spanish version of the TSRQ showed adequate psychometric properties (dimensionality and internal structure, reliability, and validity evidence regarding its relationships with other constructs) in college students. The Spanish TSRQ will provide future research aimed to understand the motivational role in college students' health behavior and well-being.