A mild biorefinery process was investigated on the microalga Nannochloropsis gaditana, to obtain an enriched fraction of water soluble proteins free from chlorophyll. After harvesting, a 100 g.L−1 solution of cells was first subjected to cell disruption by either high-pressure homogenization (HPH) or enzymatic treatment (ENZ). HPH resulted in a larger release of proteins (49%) in the aqueous phase compared to the Alcalase incubation (35%). In both cases, an ultrafiltration/diafiltration (UF/DF) was then performed on the supernatant obtained from cell disruption by testing different membrane cut-off (1000 kDa, 500 kDa and 300 kDa). After optimising the process conditions, the combination of ENZ → UF/DF ended in a larger overall yield of water soluble proteins (24.8%) in the permeate compared to the combination of HPH → UF/DF (17.4%). A gel polarization model was implemented to assess the maximum achievable concentration factor during ultrafiltration and the mass transfer coefficient related to the theoretical permeation flux rate.