Hydroxyanthracene derivatives (HADs) are plant substances produced by a variety of plant species, including different , , and species and These plants are often used in food supplements to improve bowel function. However, recently, the European Commission prohibited a number of HADs due to toxicological concerns. These HADs included aloin (aloin A and aloin B), aloe-emodin, emodin, and danthron. Most of the currently available analytical methods are restricted to the analysis of only these compounds and do not include other HADs. In this view, a multi-analyte method could be useful for both regulatory analysis and dietary intake studies. To this end, such a method, employing liquid chromatography-tandem mass spectrometry and targeting 16 different HADs, was developed and validated in this study. Limits of quantification were in the range from 0.025 mg kg to 1 mg kg. The recovery of the method was within the acceptable range of 80% to 120%, with the exception of physcion. Repeatability varied from 0.5% to 11.6%, and the range for within-laboratory reproducibility was from 3.4% to 16.3%. The expanded measurement uncertainty was below 50% for all HADs. Subsequently, 24 commercial samples of food supplements and herbal infusions sourced in Belgium were analyzed. The results indicated that although the industry put a great effort into minimizing the amount of aloin and danthron present in food supplements, more than half of the products still exceeded the maximum tolerated levels suggested for aloe-emodin and emodin.