Repeat associated non-AUG (RAN) initiated translation of FMR1 5’ UTR CGG repeats produces toxic homo-polymeric proteins that accumulate within ubiquitinated inclusions in Fragile X-associated tremor/ataxia syndrome (FXTAS) patient brains and model systems. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG codon located 5’ to the repeat. Methods to accurately measure FMRpolyG in FXTAS patients are lacking. Here we used data dependent acquisition (DDA) and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides (SIS) to identify potential signature FMRpolyG fragments in patient cells and tissues. Following immunoprecipitation enrichment, we detected FMRpolyG signature peptides by PRM in transfected cells, FXTAS human samples and patient derived stem cells, but not in controls. Surprisingly, we identified two amino-terminal peptides: Ac-MEAPLPGGVR, initiating at an ACG, and Ac-TEAPLPGGVR, initiating at a GUG, along with evidence for RAN translation initiation within the CGG repeat itself. Initiation at all three sites was upregulated in response to cellular stress, downregulated following eIF1 overexpression and dependent on the initiation factor helicase, eIF4A. Use of the two initiation sites upstream of the repeat decreased following loss of the eIF2 alternative factor eIF2A. These data demonstrate that FMRpolyG is quantifiable in human samples and that RAN translation on FMR1 initiates via similar mechanisms at both near-cognate codons and within the repeat itself through processes dependent on available initiation factors and the cellular environment.