open FILE, "96well2.txt" or die $!;
 while (<FILE>){
 chomp $_;
  @values = split('\t', $_);
  $val = @values;
  print $val;
 }
 close (FILE);
[download]

As $val = @values assigns the number of elements in the array to $val, your output confirms my suspicion, i.e. that the lines aren't tab separated, so you get only one value in @values (the entire line) from the split.

Try splitting on whitespace:

#!/usr/bin/perl
use strict;
use warnings;

print " Enter the dilution factor. \n";
chomp (my $df = <STDIN>);
open FILE, "45well.txt" or die $!;
while (<FILE>){
    chomp;
    my @values = split ' ';
    foreach my $val (@values){
        my $DNA_conc = $val * $df * 50 ;
        print "$DNA_conc\n";
    }
}
close (FILE);
[download]

The code has started to work finally.. but the problem is it is giving the output in one column and splitting all the values.

so for the line :

0.250 0.413 0.432 0.345 0.786 1.001 0.987

the output is :

125
206.5
216
172.5
393
[download]

and so on

how should i put it back together? This is the code i used :

use warnings;
print " Enter the dilution factor. \n";
chomp ($df = <STDIN>);
open FILE, "96well.txt" or die $!;
while (<FILE>){
 chomp $_;
  @values = split('\t', $_);

foreach $val (@values){
$DNA_conc = $val * $df * 50 ;
print $DNA_conc;
print"\n";
}
}
 close (FILE);
[download]