Background Differential Item Functioning (DIF) is investigated to ensure that each item displays a consistent pattern of responses irrespective of the characteristics of the respondents. Assessing DIF helps to understand the nature of instruments, to assess the quality of a measure and to interpret results. This study aimed to examine whether the items of the Quality of Life Enjoyment and Satisfaction Questionnaire-Short Form (Q-LES-Q-SF) and Short-Form 12 (SF-12) exhibit DIF. Method A total of 124 outpatients diagnosed with substance dependence participated in a cross-sectional, multicenter study. In addition to the Q-LES-Q-SF and SF-12 results, demographic data such as age, sex, type of substance dependence and education level were collected. Rasch analysis was conducted (using RUMM2020 software) to assess DIF of the Q-LES-Q-SF and SF-12 items. Results For SF-12, significant age-related uniform DIF was found in two of the 12 items, and sex-related DIF was found in one of the 12 items. All of the observed DIF effects in SF-12 were found among the mental health items. Three items showed DIF on the Q-LES-Q-SF; however, the impact of DIF item on the delta score calculation for the comparisons of self-reported health status between the groups was minimal in the SF-12 and small in the Q-LES-Q-SF. Conclusion These results indicated that no major measurement bias affects the validity of the self-reported health status assessed using the Q-LES-Q-SF or SF-12. Thus, these questionnaires are largely robust measures of self-reported health status among substance users.