Background: The active (remote) loading of drugs into nanoparticulate systems via the pH gradient technique has been proven highly successful in liposomes, as numerous formulations have reached the market. However, this is not the case for niosomes, as the full potential of this area remains largely undiscovered. The purpose of this research is to study the effect of different co-surfactants (Cremophor RH 40, Cremophor ELP and Solutol HS-15) on stabilising the niosomal membrane to enable the creation of a pH gradient. Methods: For visualisation of pH gradients, pH indicator bromocresol green (BCG) was used as a novel encapsulated model molecule to visually investigate the ability of niosomes to entrap drugs through active loading. Thereafter, the optimised BCG niosomal formulation was applied to encapsulate a therapeutic drug molecule, doxorubicin, via pH gradient active loading. Niosomes were formulated via thin-film hydration using Span 60, cholesterol, with or without co-surfactants. Thin films were hydrated with either Trizma buffer or HEPES buffer for BCG, or ammonium sulfate for doxorubicin. The niosomes’ outer membrane pH was adjusted via either the addition of HCl or citric acid in the case of BCG, or by passing the niosomes through a Sephadex G50 gel column, pre-equilibrated with PBS or Trizma buffer, in the case of doxorubicin. Results: Niosomes formulated with Span 60 and cholesterol could not be formed at acidic pH and thus could not create a pH gradient. All three co-surfactants, when added to Span 60 and cholesterol, stabilised the niosomes and enabled them to form a pH gradient. Niosomes (after size reduction) containing Solutol HS-15 showed significantly higher entrapment efficiency of BCG when compared to Cremophor RH 40 and Cremophor ELP (67.86% vs. 15.57% vs. 17.81%, respectively, with sizes of 159.6 nm, 177.9 nm and 219.1 nm, respectively). The use of HEPES buffer resulted in a higher EE of BCG compared to Trizma buffer (72.85% vs. 67.86%) and achieved a size of 283.4 nm. The Solutol HS-15 containing formulation has exhibited 68.28% EE of doxorubicin with ammonium sulfate as the inner buffer, while the external buffer was Trizma with a size of 241.1 nm after extrusion. Conclusions: Niosomal formulations containing Solutol HS-15 are highly promising for remote drug loading. The novel use of BCG for studying pH gradient and drug loading into niosomes has proved beneficial and successful.