There is a great discrepancy between the natural product output of cultured microorganisms and their bioinformatically predicted biosynthetic potential, such that most of the molecular diversity contained within microbial reservoirs has yet to be discovered. One of the primary reasons is insufficient expression of natural product biosynthetic gene clusters (BGCs) under standard laboratory conditions. Several methods have been developed to increase production from such “cryptic” BGCs. Among these, we recently implemented mass spectrometry-guided transposon mutagenesis, a forward genetic screen in which mutants that exhibit stimulated biosynthesis of cryptic metabolites, as read out by mass spectrometry, are selected from a transposon mutant library. Herein, we use Burkholderia gladioli as an example and provide guidelines for generating transposon mutant libraries, measuring metabolomic inventories through mass spectrometry, performing comparative metabolomics to prioritize cryptic natural products from the mutant library, and isolating and characterizing novel natural products elicited through mutagenesis. Application of this approach will be useful in both accessing novel natural products from cryptic BGCs and identifying genes involved in their global regulation.