Split on ':' and '.', then convert to measurement in pure-millisecond, then do a minus (assume this is always the same day)

my $t1 = "05:07:53.249";
my $t2 = "05:07:55.401";
print to_milli($t2) - to_milli($t1);

sub to_milli {
    my @components = split /[:\.]/, shift;
    return (($components[0] * 60 + $components[1]) * 60 + $components[
+2]) * 1000 + $components[3];
}
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You could convert to seconds and use the result for your calculations. The conversion is straightforward:

my ($h, $m, $s) = split /:/, $time;
my $time_in_secs = $h * 60 * 60 + $m * 60 * $s;
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Time::HiRes::tv_interval seems to work just fine for this.