Background: There are currently several second-line options for the treatment of ASTS as gemcitabine combinations, trabectedin, pazopanib, eribulin or olaratumab plus doxorubicin in cases where anthracyclins are still possible. There is an unmet need for predictive biomarkers which hinders the rational selection of the best sequence in second line. We already published the prognostic value of FAS in first line of ASTS while this study analyzes its predictive role in different second line schemes. Methods: Most relevant selection criteria for this study were having received trabectedin in 2^nd line or beyond for ASTS, progressive disease after at least one previous line for ASTS and signed CI. A TMA was set up for FAS staining (Cell Signaling) with blocks from diagnostic time. Two expert blinded pathologists reviewed and classified the cases as negative, weak or strong. Kaplan–Meier estimations were used for time-to-event variables and the log-rank test was used to compare groups. Results: A series of 198 patients accomplished selection criteria. Metastases at diagnosis occurred in 46 (24%) and median time to metastases was 18.8 months (CI 16,3; 21.3). Previous line to trabectedin consisted of gemcitabine combination 83 (42%), Doxorubicin-based 65 (33%) and others 50 (25%). Median PFS for previous and trabectedin lines were 3.5 (2.8-4.2) and 3.4 (2.8-4) months respectively. FAS positive entailed significantly better PFS for the previous trabectedin line: 4.1 (1.5-6.7) vs 3.0 (2.5-3.5) months, p = 0.01 whereas FAS positive was related with worse PFS for the trabectedin line 2.5 (2.2-2.8) vs 3.7 (2.7-4.8) months, p = 0.028. These results were more notorious for L-sarcoma cases: 7.0 (3.6-10.5) vs 4.3 (1.9-6.6) months, p = 0.017 in previous line and 2.4 (2.2-2.6) vs 6.5 (3.8-9.3) months, p < 0.001 in trabectedin. From trabectedin administration, FAS+ had significantly worse OS especially in L-sarcomas: 11.9 (5.2-18.7) vs 21.7 (12.7-30.8) months, p = 0.002. Conclusions: FAS showed predictive value in PFS and OS for trabectedin administration in ASTS. The different prognostic role of FAS across distinct lines and its relevance in L-sarcomas deserve further attention.