Then you're starting back at 0 for each $prot_string... I'm not sure that's what you want...You can also use index()

foreach $prot_string (@truncated_sequences) {
    foreach $tri_seq (@triamino_combo) {
        $i=-1;
        while(($i=index($prot_string,$tri_seq,$i)) > -1){
            $tri_freq{$tri_seq}++;
            $i+=3;
        }
        $tri_freq{$tri_seq}='0' unless defined $tri_freq{$tri_seq};
    }
}
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