An innovative, simple, fast, specific and sensitive high performance liquid chromatographic method with ultraviolet detection (HPLC/UV) was established and validated for simultaneous determination of a combination of three commonly prescribed antigout drugs namely; Colchicine (CLN), Probenecid (PRD) and Febuxostat (FBX) in dosage forms and in human urine samples. GraceSmart RP C18 column was utilized for separation of the studied mixture using isocratic mode of mobile system consisting of acetonitrile: water (55: 45, v/v; containing 0.5% v/v formic acid). Flow rate and detection wavelengths programming were designed to obtain a rapid and efficient separation. The cited drugs were separated within <9 min. The retention times (tR) were 3.597, 5.357 and 8.250 for CLN, PRD and FBX; respectively. The linearity range for all the investigated drugs was 0.040–50.0 μg/mL with detection limits of 1.94–6.48 ng/mL. Additionally, the developed method was validated according to ICH and US-FDA guidelines and was successfully applied for simultaneous estimation of the studied drugs in their pure form, and in laboratory-made mixture solution of their respective tablets. The proposed method was also used for analysis of CLN and PRD in their combined tablets and in human urine samples obtained from healthy volunteers with good recoveries (98.39–101.87%). Furthermore, the stability of CLN-PRD mixture in urine samples was studied. In conclusion, this method is suitable for quality control purposes for simultaneous analysis of these co-administered antigout drugs in their binary and ternary mixtures.