Roxithromycin is one of the most frequently used macrolide antibiotics, a safe group of antimicrobials, that acts against Gram-positive bacteria and some Gram-negative bacteria. It is sold in several countries in different dosage forms (tablets and capsules) and strengths (50, 100, 150 and 300 mg). Several analytical methods have been described to quantify roxithromycin in different matrices, such as biological and environmental samples and food. Identifying the main characteristics of the drugs and selecting appropriate analytical methods for their quantification are of paramount importance for understanding the behavior of drugs, metabolites and impurities. This review presents the physical-chemical and microbiological characteristics, properties as well as methods for quantification of roxithromycin in biological fluids, pharmaceutical preparations, food and environmental samples. HPLC coupled to various detection systems is the most used analytical technique to determine roxithromycin in these matrices. Although many analytical methods have been reported for the analysis of this drug, it is very important to include in this context a prospective view to implement new analytical technologies for the quality control of this antimicrobial that contributes to the preservation of economic and environmental impacts.