This study aims at evaluating the impact of different processes-pulsed electric fields (PEF), pressurized liquid extraction (PLE) and a multistep process combining PEF + PLE on the yield of antioxidant compounds (protein, polyphenols, chlorophyll a, chlorophyll b, and carotenoids) from Spirulina. Firstly, the effects of PEF or PLE treatment on the extraction yield of Spirulina biomolecules were evaluated. To further increase the extraction yield, PEF + PLE was used, as an innovative extraction approach. The results showed that PEF + PLE greatly improved the extraction yield compared with the PEF or PLE treatments alone. Compared with Folch extraction (conventional control technique), PEF + PLE significantly (P < 0.05) shortened the extraction time (−165 min) and increased the protein, polyphenol, chlorophyll a and antioxidant capacity values of Spirulina extracts by 1328%, 979%, 11% and 47% respectively. Furthermore, Triple TOF-LC-MS-MS results showed that PEF + PLE increased both the type and content of phenolic compounds. The above results were attributed to PEF-induced damage on Spirulina helical structure, which was verified by fluorescence and scanning electron microscopy.