Reversed phase high-performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC)-spectrodensitometric methods have been developed and validated for the separation and quantitation of two binary mixtures: Ofloxacin (OFX) and dexamethasone (DXM) in eye preparation; ciprofloxacin hydrochloride (CIP) and hydrocortisone (HYD) in ear preparation. The linearity ranges of RP-HPLC methods were found to be (2.5–45 μg mL−1) for OFX, (2.5–50 μg mL−1) for DXM and (1–8 μg mL−1) for both CIP and HYD. The percentage recoveries/relative standard deviation (RSD) were found to be 100.36/1.38, 100.13/1.49, 99.98/0.61 and 100.28/1.27, respectively. The linearity ranges of TLC-spectrodensitometric methods were found to be (0.5–2 μg band−1), (0.5–3.5 μg band−1), (0.2–1.6 μg band−1), and (0.6–2 μg band−1) for OFX, DXM, CIP, and HYD, respectively. The percentage recoveries/RSD were found to be 99.98/1.06, 99.93/1.18, 99.74/1.27, and 99.94/1.54, respectively. A comparative study was conducted to show the advantages of the proposed methods which showed that the TLC-spectrodensitometric methods were simpler, more sensitive, and economic, while RP-HPLC methods were more precise and robust. The methods were validated in compliance with the ICH guidelines and were successfully applied for determination of the selected drugs in their laboratory-prepared mixtures and commercial dosage forms.