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Background: The development of innovative approaches in drug analysis is a challenging task for medicine, pharmacy, and engineering sciences. For instance, the requirement of proper dosage forms in releasing active ingredients is crucial. It is essential to analyze drugs in biological liquids for early diagnosis and treatment purposes. Drug analysis is also of great importance to control the quality of pharmaceutical products, test their efficacy, and develop novel drug formulation. The present review is aimed to highlight the most recent spectrophotometric approaches applied to analyze various classes of drugs in biological media and/or dosage forms. Methods: Direct and derivative UV/UV-Vis spectrophotometry and a combination of various techniques with spectrophotometry, such as injection analysis and chemometrics, have most widely been applied in the analysis of dosage forms. In addition, emerging technologies, such as UV imaging allows to obtain the distribution of drug concentration in time-resolved 2D images based on UV light absorption, utilization of nanotechnology, self-assembled nanomaterials, and aptamer-based nanoparticles have gained interests to investigate drug assays and to quantify the proper drug release. Results: Due to their high versatility, ease of application, low cost, and fast response, spectrophotometric methods are one of the most preferable methods providing high accuracy and precision with a wide linear range in drug analysis. Conclusion: Selected examples demonstrating the applicability of spectrophotometric methods in pharmaceutical assays in this review might contribute to the overall importance of the analytical test used in modern pharmaceutical analysis.