An isolation method based on protection-deprotection, with possible applicability to other unstable natural products, is described for tunichromes. Isolation of labile natural products is often hindered as they lose their stabilizing microenvironment (e.g., antioxidants, membranes) during isolation. Tunichromes An-1-3 (polyphenolic tripeptides with enamide moieties) from vanadium-assimilating tunicates decompose upon air exposure and precipitate with VV/VIV; initial isolation required argon, anhydrous conditions, lyophilization, and centrifugal partition chromatography (CPC), with 80% loss during reverse phase HPLC purification. Tunichromes Mm-1/2 from Fe-assimilating tunicates were acetylated, but free forms could not be regenerated from peracetates, making this unsuitable for biological studies. Recent total syntheses used t-butyldimethylsilyl (TBDMS, for hydroxy groups) and butoxycarbonyl (Boc, for amino groups) protection, yielding free tunichromes after deprotection. Applying these strategies to tunicate blood enabled efficient, simplified isolation and yielded additional unknown tunichrome-related pigments. Lyophilized blood pellets were ground with anhydrous Na2SO4, treated with TBDMSCl/imidazole (CH2Cl2) to protect hydroxy groups (converting polar, unstable tunichromes to stable, nonpolar derivatives), then with (Boc)2O to protect amino groups. Protected An-1, An-2, and An-3 were separated via preparative TLC and flash chromatography. Deprotection (trifluoroacetic acid [TFA] for Boc, 48% aqueous HF-pyridine for TBDMS) and fractional precipitation afforded natural An-1, confirmed by 1H NMR, UV, and mass spectra. This method facilitates separation of closely related tunichromes (An-1: Rf 0.44; An-2: 0.4; An-3: 0.33 via preparative TLC) and enables isolation from stored blood pellets. Four new bright yellow pigments (UV absorption similar to tunichromes) were also isolated, with characterization ongoing. The method is applicable where peracetate hydrolysis or demethylation is problematic (e.g., tunichromes) and to other unstable natural products containing hydroxy and/or amino groups.