With the global drive for faster, more environmentally friendly separation techniques, the aim of this research was to demonstrate the potential of Supercritical Fluid Chromatography (SFC) as a viable alternative or complementary technique to High Performance Liquid Chromatography (HPLC) in the highly regulated world of the Quality Control (QC) laboratory. SFC methods capable of meeting QC method performance expectations in accordance with current guidance were therefore developed and validated under current International Conference of Harmonisation (ICH) guidance. Firstly, an enantioselective pSFC method was developed and validated to meet the current European Pharmacopoeia requirements of a limit test for the determination of S-timolol maleate enantiomeric purity in timolol maleate drug substance. The newly developed pSFC method achieved a resolution of 2.0 within 5 min, representing a 3-fold reduction in run-time and an 11-fold reduction in solvent consumption relative to the normal phase HPLC method described in the European Pharmacopoeia. Secondly, a stability-indicating Reversed Phase (RP-HPLC) method was developed and validated for the determination of prednisolone acetate (PAC) and eight selected PAC impurities and degradation products in an ophthalmic suspension using a superficially porous “core-shell” stationary phase. Using an Agilent Poroshell column with step gradient elution, all peaks of interest were eluted in 33 min with resolution of 1.5 between the critical pairs. With core-shell stationary phases being considered the most efficient technology currently available for packed column HPLC applications, this RP-HPLC method was developed to enable a direct comparison to be made between RP-HPLC and SFC in terms of orthogonality, efficiency, selectivity, sensitivity and reproducibility. Finally, an orthogonal achiral pSFC method was developed and validated for the same PAC sample described above. For the pSFC method, validation was carried out using the total error approach to generate accuracy profiles for two regression models, based on β-expectation tolerance intervals. Successful completion of the method validation demonstrated that the new pSFC method was a viable complementary or alternative to the previously developed RP-HPLC method for use in the highly regulated QC laboratory environment.