An N × n matrix on q symbols is called {w 1 , . . . , w t }-separating if for arbitrary t pairwise disjoint column sets C 1 , . . . , C t with |C i | = w i for 1 ≤ i ≤ t, there exists a row f such that f (C 1 ), . . . , f (C t ) are also pairwise disjoint, where f (C i ) denotes the collection of components of C i restricted to row f . Given integers N, q and w 1 , . . . , w t , denote by C(N, q, {w 1 , . . . , w t }) the maximal n such that a corresponding matrix does exist. The determination of C(N, q, {w 1 , . . . , w t }) has received remarkable attentions during the recent years. The main purpose of this paper is to introduce two novel methodologies to attack the upper bound of C(N, q, {w 1 , . . . , w t }). The first one is a combination of the famous graph removal lemma in extremal graph theory and a Johnson-type recursive inequality in coding theory, and the second one is the probabilistic method. As a consequence, we obtain several intriguing upper bounds for some parameters of C(N, q, {w 1 , . . . , w t }), which significantly improve the previously known results.