Ciguatoxins (CTXs) are polyether marine biotoxins that can cause ciguatera poisoning (CP) after the consumption of fish or invertebrates containing sub ppb levels; concentrations that present a challenge for current extraction and analysis methods. Here, a newly developed and (partly) validated single-day extraction protocol is presented. First, the fish sample is broken-down by enzymatic digestion, followed by extraction and extract clean-up by defatting and two solid-phase extractions. Final extracts were investigated using two different CTX-analysis methods; an in vitro cytotoxicity assay (N2a-assay) and by LC-MS/MS. Validation was performed for both fillet and freeze-dried samples of snapper, parrotfish, and grouper spiked with CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, and CTX3C. Based on recovery rates (35–88%) and matrix effects (66–116%) determined by LC-MS/MS, the enzyme protocol is applicable to various matrices. The protocol was applied to naturally contaminated fish tissue (Lutjanus bohar) obtained during a CP incident in Germany. Several potential CTX congeners were identified by a two-tier LC-MS/MS approach (screening of sodium adducts, high-resolution or low-resolution confirmation via ammonium adducts). Inclusion of >30 known CTX congeners into the LC-MS/MS methods and single-day sample preparation make the method suitable for analysis of ciguatera suspect samples at sub ppb levels also with undisclosed CTX profiles.