This review summarizes the literature data reported from 2000 up to the present on the development of various electrochemical (voltammetric, amperometric, potentiometric and photoelectrochemical), optical (UV-Vis and IR) and luminescence (chemiluminescence and fluorescence) methods and the corresponding sensors for rifamycin antibiotics analysis. The discussion is focused mainly on the foremost compound of this class of macrocyclic drugs, namely rifampicin (RIF), which is a first-line antituberculosis agent derived from rifampicin SV (RSV). RIF and RSV also have excellent therapeutic action in the treatment of other bacterial infectious diseases. Due to the side-effects (e.g., prevalence of drug-resistant bacteria, hepatotoxicity) of long-term RIF intake, drug monitoring in patients is of real importance in establishing the optimum RIF dose, and therefore, reliable, rapid and simple methods of analysis are required. Based on the studies published on this topic in the last two decades, the sensing principles, some examples of sensors preparation procedures, as well as the performance characteristics (linear range, limits of detection and quantification) of analytical methods for RIF determination, are compared and correlated, critically emphasizing their benefits and limitations. Examples of spectrometric and electrochemical investigations of RIF interaction with biologically important molecules are also presented.