Complete and uniform application of postchromatographic derivatization reagents to the layer is critical for sensitive detection and accurate and precise quantification of separated analytes lacking a chromophore in high performance thin layer chromatography (HPTLC). In this research, standards representing the five major neutral lipid classes were separated on silica gel HPTLC plates by development with petroleum ether-diethyl ether-glacial acetic acid (80:20:1) and detected as blue zones using ethanolic phosphomolybdic acid (PMA) reagent. Qualitative and quantitative results for the lipids were compared after applying PMA by the two most widely used methods, manual spraying and dipping, and with the new manual Derivapress device, followed by heating of the plate on a plate heater. The application methods were compared in terms of accuracy, precision, visible and densitometric limit of detection, calibration curves, cost effectiveness, ease of use, and safety. Samples were applied to the layer using a spray-on band applicator, and a densitometer was used for instrumental detection and quantification at 610 nm. Results showed that dipping is the best PMA application method for quantitative analysis and that spraying is best for high sensitivity qualitative analysis. The Derivapress proved to be simple and economical for application of PMA in qualitative and quantitative analysis of neutral lipids. These comparative procedures can serve as a general model for optimization of the detection step in any analyte-layer-mobile phase system using all possible postchromagraphic derivatization reagent application methods: manual and instrumental spraying, manual and instrumental dipping, modified commercial printer, or Derivapress.