With the increased production of new fixed combinations and cheaper generic drugs, the need for a single, sensitive and reliable analytical method is hugely evoked to cover the versatile and prerequisite tests of in-vitro and in-vivo bioequivalence studies. A new fixed dose combination, containing dutasteride (DUT) and silodosin (SLD), has been recently launched for relieving moderate and severe symptoms of benign prostatic hyperplasia. Consequently, this concurrent study was dedicated to present, for the first time, eco-friendly high-performance liquid chromatographic (HPLC) methods coupled with dual-mode detection, for the simultaneous quantification of both drugs in versatile real samples. The optimized chromatographic separation was achieved on a Waters XBridge® (150×4.6 mm, 5 μm) C18 analytical column with a mobile phase composed of methanol & phosphate buffer pH 6.0 at ratio (77: 23, v/v) and pumped at 1.0 mL/min flow rate. In the first HPLC method, drug quantification was performed at 245 nm achieving linearity range of 3–120 µg/mL, while the second one made good use of the unique features of both drugs in possessing native fluorescence for more sensitive linearity ranges (50–1000 ng/mL for DUT & 80–2500 ng/mL for SLD). Both methods were validated and successfully applied for assay dosage forms as well as testing content uniformity. In addition, the capability of the proposed HPLC-fluorometric method in detecting drugs in nano-gram scale paved the way not only for studying their dissolution behavior in various dissolution media but also analyzing spiked human plasma samples extracted by liquid-liquid extraction. Finally, sustainability of the proposed methods was clearly declared, in comparison to the official ones, via environmental, health and safety (EHS) tool and analytical greenness (AGREE) software.