The present study aimed at the development and validation of a first-derivative ultraviolet (UV) spectrophotometric method for the estimation of Lopinavir in formulation as well as in bulk drug. The reported methods of analysis include chromatographic methods such as high-performance liquid chromatography using a UV detector. Chromatographic methods are tedious, time consuming and are not suitable for routine analysis. However, UV-based methods are fast, simple and economical. Thus, these methods are preferred for routine analysis. The absorption scan of Lopinavir in the UV region does not exhibit any sharp peak. However, the first-derivative spectrum showed an absorption minima at 220 nm, which was selected as the analytical wavelength. Thus, a first-derivative method was developed and validated for the routine analysis of Lopinavir. The method was found to be simple, sensitive and selective, as evidenced by non-interference from the excipients and ritonavir present in the tablet formulation. The linearity was observed in the range of 5 to 35 μg/ml. The method was found to have a high degree of accuracy and precision, both inter- and intra-day. The limit of detection and limit of quantitation were found to be 0.844 and 2.558 μg/ml, respectively.